Light-sensitive ion-passing molecules

ABSTRACT

The invention provides polynucleotides and methods for expressing light-activated proteins in animal cells and altering an action potential of the cells by optical stimulation. The invention also provides animal cells and non-human animals comprising cells expressing the light-activated proteins.

RELATED PATENT DOCUMENT

This patent document claims the benefit, under 35 U.S.C. §119(e), ofU.S. Provisional Patent Application Ser. No. 61/314,969 filed on Mar.17, 2010, and entitled “Light-Sensitive Ion-Passing Molecules;” thispatent document and the Appendices filed in the underlying provisionalapplication are fully incorporated herein by reference.

OVERVIEW AND SUMMARY

Aspects of the present disclosure relate generally to systems andapproaches for stimulating target cells, and more particularly to usingoptics to stimulate the target cells. The stimulation of various cellsof the body has been used to produce a number of beneficial effects. Onemethod of stimulation involves the use of electrodes to introduce anexternally generated signal into cells. One problem faced byelectrode-based brain stimulation techniques is the distributed natureof neurons responsible for a given mental process. Conversely, differenttypes of neurons reside close to one another such that only certaincells in a given region of the brain are activated while performing aspecific task. Alternatively stated, not only do heterogeneous nervetracts move in parallel through tight spatial confines, but the cellbodies themselves may exist in mixed, sparsely embedded configurations.This distributed manner of processing seems to defy the best attempts tounderstand canonical order within the CNS, and makes neuromodulation adifficult therapeutic endeavor. This architecture of the brain poses aproblem for electrode-based stimulation because electrodes arerelatively indiscriminate with regards to the underlying physiology ofthe neurons that they stimulate. Instead, physical proximity of theelectrode poles to the neuron is often the single largest determiningfactor as to which neurons will be stimulated. Accordingly, it isgenerally not feasible to absolutely restrict stimulation to a singleclass of neuron using electrodes.

Another issue with the use of electrodes for stimulation is that becauseelectrode placement dictates which neurons will be stimulated,mechanical stability is frequently inadequate, and results in leadmigration of the electrodes from the targeted area. Moreover, after aperiod of time within the body, electrode leads frequently becomeencapsulated with glial cells, raising the effective electricalresistance of the electrodes, and hence the electrical power deliveryrequired to reach targeted cells. Compensatory increases in voltage,frequency or pulse width, however, may spread the electrical current andincrease the unintended stimulation of additional cells.

Another method of stimulus uses photosensitive bio-molecular structuresto stimulate target cells in response to light. For instance, lightactivated proteins can be used to control the flow of ions through cellmembranes. By facilitating or inhibiting the flow of positive ornegative ions through cell membranes, the cell can be brieflydepolarized, depolarized and maintained in that state, orhyperpolarized. Neurons are an example of a type of cell that uses theelectrical currents created by depolarization to generate communicationsignals (i.e., nerve impulses). Other electrically excitable cellsinclude skeletal muscle, cardiac muscle, and endocrine cells. Neuronsuse rapid depolarization to transmit signals throughout the body and forvarious purposes, such as motor control (e.g., muscle contractions),sensory responses (e.g., touch, hearing, and other senses) andcomputational functions (e.g., brain functions). Thus, the control ofthe depolarization of cells can be beneficial for a number of differentpurposes, including (but not limited to) psychological therapy, musclecontrol and sensory functions.

Various aspects of the present invention are directed toward ablue-light sensing opsin capable of inhibiting neural activity. Theopsin comes from cryptophytes Guillardia theta (G. theta). The opsin ofinterest is the third opsin isolated from G. theta, and is abbreviatedGtR3. GtR3 is capable of mediating a hyperpolarizing current whenilluminated with light. Characterization of the action spectra for GtR3suggests that the absorption maxima are around 490 nm, and GtR3 is notactivated by yellow light.

Various aspects of the present invention are directed to a blue-lightsensing channelrhodopsin capable of exciting neural activity. Thechannelrhodopsin is derived from Dunaliella salina. The channelrhodopsinof interest is abbreviated as DChR. DChR can be heterologously expressedin mammalian neurons and mediates a robust depolarizing current whenilluminated with blue light. The action maxima for DChR are around 500nm.

Consistent with an embodiment of the present disclosure, an inhibitorycurrent flow is created by engineering a protein derived from Guillardiatheta that responds to light by producing an inhibitory current todissuade depolarization of a neuron. The protein is delivered to aneuron of interest and the neuron is exposed to light.

Consistent with another embodiment of the present disclosure, a methodof optical stimulation of a cell expressing a GtR3 proton pump comprisesproviding a sequence of stimuli to the cell, each stimulus increasingthe probability of a depolarizing event occurring in the cell. Light isprovided to the cell to activate the expression of the GtR3 proton pump,thereby decreasing the probability of the depolarizing event occurringin the cell. In certain specific embodiments the light provided is inthe blue light spectrum.

Consistent with another embodiment of the present disclosure, a systemfor controlling an action potential of a neuron or other cell in vivo isdisclosed. The system comprises a delivery device that introduces aprotein responsive to blue light to the neuron or cell. The proteinresponsive to blue light produces an inhibitory current in response toblue light. The system includes a blue light source that generates lightfor stimulation of the blue light responsive protein and a controldevice that controls the generation of light by the light source.

Consistent with another embodiment of the present disclosure, a methodfor providing a light responsive protein for mammalian expression isdisclosed. A light responsive protein is isolated from G. theta. Theisolated protein has a C-terminus and an N-terminus. An endoplasmicreticulum (ER) export signal is added to the C-terminus of the isolatedprotein to create an enhanced light responsive protein. The enhancedprotein is placed in an empty virus vector for delivery to a cell ofinterest. The virus vector with the enhanced protein is then deliveredto the cell of interest.

Consistent with an embodiment of the present disclosure an animal cellis provided. The animal cell includes an integrated exogenous moleculewhich expresses a proton pump responsive to blue light. The exogenousmolecule is derived from G. theta. In certain embodiments the animalcell is a neural cell. The animal cell may also be a muscle cell or acell line, for example.

The above discussion of the present disclosure is not intended todescribe each illustrated embodiment or every implementation of thepresent invention. The figures and detailed description that follow moreparticularly exemplify these embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more completely understood in consideration of thedetailed description of various embodiments of the invention thatfollows in connection with the accompanying drawings as follows:

FIG. 1A shows an ion pump in an organelle membrane;

FIG. 1B shows an ion pump in a cell membrane;

FIG. 2A shows cell populations expressing combinations of lightresponsive proteins;

FIG. 2B shows a stimulus profile for use with certain embodiments inwhich two or more light responsive proteins are introduced into the samecell population;

FIG. 3 shows a block diagram of a system for stimulating target cells,according to an example embodiment of the present invention;

FIG. 4 shows a block diagram of an implantable device for stimulatingtarget cells, according to an example embodiment of the presentinvention;

FIG. 5 shows a block diagram of an implantable device, according to anexample embodiment of the present invention;

FIG. 6A shows a block diagram of an implantable device, according to anexample embodiment of the present invention;

FIG. 6B shows a circuit diagram corresponding to the block diagram ofFIG. 6A, according to an example embodiment of the present invention;

FIG. 7A and FIG. 7B show a diagram of a mesh for containingphotosensitive bio-molecules, according to an example embodiment of thepresent invention;

FIG. 8A and FIG. 8B show a diagram of a viral matrix, according to anexample embodiment of the present invention;

FIG. 9 shows a circuit diagram of a circuit that produces light inresponse to a magnetic field, according to an example embodiment of thepresent invention;

FIGS. 10A, 10B and 10C show a block diagram and circuits for theproduction of light in response to a RF signal, according to an exampleembodiment of the present invention;

FIG. 11A and FIG. 11B each show a diagram of a fiber-optic device,according to an example embodiment of the present invention;

FIGS. 12A, 12B, 12C and 12D depict various stages in the production of aphotosensitive biological portion, according to an example embodiment ofthe present invention; and

FIG. 13 shows an implantation device, according to an example embodimentof the present invention;

FIG. 14A and FIG. 14B show a diagram for another implantation device,according to an example embodiment of the present invention;

FIG. 15 depicts an arrangement with multiple light sources, according toan example embodiment of the present invention;

FIG. 16 shows a system for controlling electrical properties of one ormore cells in vivo, according to an example embodiment of the presentinvention;

FIG. 17 shows a system for controlling electrical properties of one ormore cells in vivo, according to an example embodiment of the presentinvention

FIG. 18A shows a block diagram of a system for optical drug screening,according to an example embodiment of the present invention;

FIG. 18B shows a specific system diagram of a large-format,quasi-automated system for drug screening in accordance with the presentmethodology, according to an example embodiment of the presentinvention;

FIG. 19 shows a system diagram of a small-format, fully automated drugscreening system which operates in accordance with the inventedmethodology, according to an example embodiment of the presentinvention;

FIG. 20A depicts the workings of an example of emitter/detector units,according to an example embodiment of the present invention;

FIG. 20B depicts the workings of another embodiment of emitter/detectorunits, according to an example embodiment of the present invention;

FIGS. 21A and 21B depict an electronic circuit mechanism for activatingthe LED emitters used within the emitter/detector units, according to anexample embodiment of the present invention;

FIG. 22 shows a timeline for a sequence of events in the context of anexample screening process, according to an example embodiment of thepresent invention;

FIG. 23 illustrates an example of a layout of cell and drug sampleswithin the wells of a well-plate, according to an example embodiment ofthe present invention; and

FIG. 24 illustrates the context in which the disclosed invention may beemployed within a larger system that facilitates high-throughput drugscreening, according to an example embodiment of the present invention.

FIG. 25D shows representative photocurrent traces showing in cellstransduced with eNpHR3.0 and cells transduced with eNpHR2.0, and summaryplots thereof;

FIG. 25E shows representative hyperpolarization voltage traces showingin cells transduced with eNpHR3.0 and cells transduced with eNpHR2.0,and summary plots thereof;

FIG. 26B shows a model of trans-synaptic gene activation byWGA-Cre-fusion. The schematic depicts two injection sites (one withWGA-Cre-fusion gene and another with Cre-dependent opsin virus) and longrange projections; Cre can be trans-synaptically delivered fromtransduced cells to activate distant gene expression only insynaptically connected neurons that have received the Cre-dependentvirus.

FIG. 26C shows construct design for the WGA-Cre and Cre-dependent AAVvectors optimized with mammalian codons;

FIG. 27A shows that six hundred thirty nanometer light evokes robustphotocurrents in neurons transduced with eNpHR3.0 (representativevoltage clamp trace at left). Summary plot comparing eNpHR2.0- andeNpHR3.0-expressing neurons (at right); eNpHR2.0, 42.7±4.5 pA; eNpHR3.0,239.4±28.7 pA; unpaired t test p=0.00004; n=10).

FIG. 27B shows that six hundred thirty nanometer illumination evokedrobust hyperpolarization (representative voltage clamp trace at left).Summary plot comparing eNpHR2.0- and eNpHR3.0-expressing neurons(right); 15.6±3.2 mV for eNpHR2.0 and 43.3±6.1 mV for eNpHR3.0; unpairedt test p=0.00116; n=10).

FIG. 27C shows a summary of outward photocurrents evoked by differentwavelengths of red and far-red/infrared border illumination: 630 nm,239.4±28.7 pA (left, n=10); 660 nm, 120.5±16.7 pA (middle, n=4); and 680nm: 76.3±9.1 pA (right, n=4). Power density: 3.5 mW/mm² (630 nm) and 7mW/mm² (660 nm, 680 nm).

FIG. 27D shows that illumination with red and far-red/infrared borderlight inhibited spiking induced by current injection in neuronsexpressing eNpHR3.0. Typical current-clamp traces show opticalinhibition at 630 nm (top left), 660 nm (top right), and 680 nm(bottom). Power density: 3.5 mW/mm² (630 nm) and 7 mW/mm² (660 nm, 680nm).

FIG. 27G shows that blue light (445 nm, 5 ms pulses) drove spiking at 20Hz (left) and 10 Hz (right), while simultaneous application of yellowlight (590 nm) inhibited spikes.

FIG. 27F shows activation spectrums for eNPAC, ChR2 (H134R), andeNpHR3.0 alone;

FIG. 28B shows five hundred sixty nanometer light induced outwardphotocurrents in eBR cells, and the corresponding sample trace involtage clamp;

FIG. 28C shows five hundred sixty nanometer light inducedhyperpolarizations in eBR cells, and the corresponding sample trace incurrent clamp;

FIG. 29A shows general subcellular targeting strategies for adaptingmicrobial opsin genes to metazoan intact-systems biology;

FIG. 29B shows refinement of targeting at the tissue and subcellularlevels;

FIG. 30A shows stability and recovery of potent photocurrents in cellsexpressing eNpHR3.0 when exposed to pairs of 10 second long yellow lightpulses separated in time by: 2.5 seconds, 5 seconds, 10 seconds, and 20seconds;

FIG. 30B shows a timecourse of eNpHR3.0 normalized photocurrents forlong-term continuous light exposure;

FIG. 30C shows stability of outward current of eNpHR3.0 over greaterthan 10 minutes; and

FIG. 31C shows sample current clamp and voltage clamp traces and summarydata of GtR3 function under 472 nm light.

While the invention is amenable to various modifications and alternativeforms, examples thereof have been shown by way of example in thedrawings and will be described in detail. It should be understood,however, that the intention is not to limit the invention to theparticular embodiments shown and/or described. On the contrary, theintention is to cover all modifications, equivalents, and alternativesfalling within the spirit and scope of the invention.

DETAILED DESCRIPTION

The present invention is believed to be useful for facilitatingpractical application of a variety of photosensitive bio-molecularstructures, and the invention has been found to be particularly suitedfor use in arrangements and methods dealing with cellular membranevoltage control and stimulation. While the present invention is notnecessarily limited to such applications, various aspects of theinvention may be appreciated through a discussing of various examplesusing this context.

As used herein, stimulation of a target cell is generally used todescribe modification of properties of the cell. For instance, thestimulus of a target cell may result in a change in the properties ofthe cell membrane that can lead to the depolarization or polarization ofthe target cell. In a particular instance, the target cell is a neuronand the stimulus affects the transmission of impulses by facilitating orinhibiting the generation of impulses by the neuron.

Consistent with one example embodiment of the present invention, alight-responsive protein is engineered in a cell. The protein affects aflow of ions (anions, cations or protons) across the cell membrane inresponse to light. This change in ion flow creates a correspondingchange in the electrical properties of the cells including, for example,the voltage and current flow across the cell membrane. In one instance,the protein functions in vivo using an endogenous cofactor to modify ionflow across the cell membrane. In another instance, the protein changesthe voltage across the cell membrane so as to dissuade or encourageaction potential firing in the cell. In yet another instance, theprotein is capable of changing the electrical properties of the cellwithin several milliseconds of the light being introduced.

An inhibitory protein dissuades firing of the action potential by movingthe potential of the cell away from the action potential trigger levelfor the cell. In many neurons, this means that the protein increases thenegative voltage seen across the cell membrane. In a specific instance,the protein acts as a proton pump that actively transfers protons out ofthe cell. In this manner, the protein generates an inhibitory currentacross the cell membrane. More specifically, the protein responds tolight by lowering the voltage across the cell, thereby decreasing theprobability that an action potential or depolarization will occur.

Certain aspects of the present invention are based on the identificationand development of a molecule/protein that functions as a proton pump.This proton pump is derived from the cryptophyte Guillardia theta (G.theta) and has been developed for expression in target cells. In certainmore specific embodiments the cell is a neuron. The engineered protein,GtR3, responds to blue light by producing an inhibitory current todissuade depolarization of the cell. When expressed in neural cells theproton pump (GtR3) can be used to inhibit neural activity in response toblue light stimulation. The GtR3 pump responds to optical stimulus bycreating an inhibitory current across the neural membrane. This currentinhibits action potentials while the modified cell is exposed to (blue)light.

The present disclosure also provides for the modification oflight-activated proteins expressed in a cell by the addition of one ormore amino acid sequence motifs which enhance transport to the plasmamembranes of mammalian cells. Light-activated proteins derived fromevolutionarily simpler organisms may not be expressed or tolerated bymammalian cells or may exhibit impaired subcellular localization whenexpressed at high levels in mammalian cells. Consequently, in someembodiments, the light-activated protein expressed in a cell is fused toone or more amino acid sequence motifs selected from the groupconsisting of a signal peptide, an endoplasmic reticulum (ER) exportsignal, a membrane trafficking signal, and an N-terminal golgi exportsignal. The one or more amino acid sequence motifs which enhancelight-activated protein transport to the plasma membranes of mammaliancells can be fused to the N-terminus, the C-terminus, or to both the N-and C-terminal ends of the light-activated protein. Optionally, thelight-activated protein and the one or more amino acid sequence motifsmay be separated by a linker. Additional protein motifs which canenhance light-activated protein transport to the plasma membrane of acell are described in U.S. patent application Ser. No. 12/041,628 whichis incorporated herein in its entirety.

The present disclosure additionally provides for light-activatedproteins which contain amino acid substitutions, deletions, andinsertions in the amino acid sequence of a native light-activatedprotein (such as, but not limited to, native GtR3, NpHR, DChR, and BR).Light-activated proteins include those in which one or more amino acidresidues have undergone an amino acid substitution while retaining theability to respond to light and the ability to control the polarizationstate of a plasma membrane. For example, light-activated proteins can bemade by substituting one or more amino acids into the native or wildtype amino acid sequence of the protein. In some embodiments, theinvention includes proteins comprising altered amino acid sequences incomparison with a native amino acid sequence, wherein the alteredlight-activated protein retains the characteristic light-activatednature and/or the ability to regulate ion flow across plasma membranesof the precursor protein but may have altered properties in somespecific aspects (for example, an increased or decreased sensitivity tolight, an increased or decreased sensitivity to particular wavelengthsof light, and/or an increased or decreased ability to regulate thepolarization state of the plasma membrane of a mammalian cell, ascompared to the native protein) Amino acid substitutions in a nativeprotein sequence may be conservative or non-conservative and suchsubstituted amino acid residues may or may not be one encoded by thegenetic code. The standard twenty amino acid “alphabet” is divided intochemical families based on chemical properties of their side chains.These families include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and sidw chains having aromatic groups (e.g.,tyrosine, phenylalanine, tryptophan, histidine). A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a chemically similar side chain (i.e.,replacing an amino acid possessing a basic side chain with another aminoacid with a basic side chain). A “non-conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a chemically different side chain (i.e.,replacing an amino acid having a basic side chain with an amino acidhaving an aromatic side chain).

Certain aspects of the present invention are directed to an animal cellexpressing the GtR3 molecule. In this manner, the animal cell includesan integrated exogenous molecule which expresses a proton pumpresponsive to blue light. In certain non-limiting embodiments the animalcell can be a neural cell, a muscle cell, a rod or cone cell or a cellline. In some embodiments, the animal cell is a mammalian cell.

Provided herein is an animal cell comprising a light-activated proteinexpressed on the cell membrane, wherein the protein is responsive toblue light and is derived from Guillardia theta, wherein the protein iscapable of mediating a hyperpolarizing current in the cell when the cellis illuminated with light. In some embodiments the light has awavelength between about 450 and 495 nm. In some embodiments, the lighthas a wavelength about 490 nm. In some embodiments, the light-activatedprotein comprises an amino acid sequence at least 95% identical to thesequence shown in SEQ ID NO: 1. In some embodiments, the light-activatedprotein comprises an amino acid sequence at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown inSEQ ID NO: 1. In some embodiments, the light-activated protein comprisessubstitutions, deletions, and/or insertions introduced into a nativeamino acid sequence to increase or decrease sensitivity to light,increase or decrease sensitivity to particular wavelengths of light,and/or increase or decrease the ability of the light-activated proteinto regulate the polarization state of the plasma membrane of the cell.In some embodiments, the light-activated protein contains one or moreconservative amino acid substitutions. In some embodiments, thelight-activated protein contains one or more non-conservative amino acidsubstitutions. The light-activated protein comprising substitutions,deletions, and/or insertions introduced into the native amino acidsequence suitably retains the ability to hyperpolarize the plasmamembrane of a neuronal cell in response to light.

Also provided herein is an animal cell comprising a light-activatedprotein expressed on the cell membrane, wherein the protein comprises acore amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 1and an endoplasmic reticulum (ER) export signal. The ER export signalmay be fused to the C-terminus of the core amino acid sequence or may befused to the N-terminus of the core amino acid sequence. In someembodiments, the ER export signal is linked to the core amino acidsequence by a linker. The linker can comprise any of 5, 10, 20, 30, 40,50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 aminoacids in length. The linker may further comprise a fluorescent protein,for example, but not limited to, a yellow fluorescent protein, a redfluorescent protein, a green fluorescent protein, or a cyan fluorescentprotein. In some embodiments, the ER export signal comprises the aminoacid sequence FXYENE, where X can be any amino acid. In someembodiments, the ER export signal comprises the amino acid sequenceVXXSL, where X can be any amino acid. In some embodiments, the ER exportsignal comprises the amino acid sequence FCYENEV.

Also provided herein is an animal cell comprising a light-activatedprotein expressed on the cell membrane, wherein the protein comprises acore amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 1and a signal peptide (e.g., which enhances transport to the plasmamembrane). The signal peptide may be fused to the C-terminus of the coreamino acid sequence or may be fused to the N-terminus of the core aminoacid sequence. In some embodiments, the signal peptide is linked to thecore amino acid sequence by a linker. The linker can comprise any of 5,10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,400, or 500 amino acids in length. The linker may further comprise afluorescent protein, for example, but not limited to, a yellowfluorescent protein, a red fluorescent protein, a green fluorescentprotein, or a cyan fluorescent protein. In some embodiments, the signalpeptide comprises the amino acid sequenceMDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNG. In some embodiments,other signal peptides (such as signal peptides from other opsins) may beused.

Also provided herein is an animal cell comprising a light-activatedprotein expressed on the cell membrane, wherein the protein comprises acore amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 1and a trafficking signal (e.g., which enhances transport to the plasmamembrane). The signal peptide may be fused to the C-terminus of the coreamino acid sequence or may be fused to the N-terminus of the core aminoacid sequence. In some embodiments, the signal peptide is linked to thecore amino acid sequence by a linker. The linker can comprise any of 5,10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,400, or 500 amino acids in length. The linker may further comprise afluorescent protein, for example, but not limited to, a yellowfluorescent protein, a red fluorescent protein, a green fluorescentprotein, or a cyan fluorescent protein. In some embodiments, thetrafficking signal is derived from the amino acid sequence of the humaninward rectifier potassium channel Kir2.1. In some embodiments, thetrafficking signal comprises the amino acid sequence K S R I T S E G E YI P L D Q I D I N V.

Also provided herein is an animal cell comprising a light-activatedprotein expressed on the cell membrane, wherein the protein comprises acore amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 1and two or more amino acid sequence motifs which enhance transport tothe plasma membranes of mammalian cells selected from the groupconsisting of a signal peptide, an ER export signal, and a membranetrafficking signal. In some embodiments, the light activated proteincomprises an N-terminal signal peptide and a C-terminal ER exportsignal. In some embodiments, the light activated protein comprises anN-terminal signal peptide and a C-terminal trafficking signal. In someembodiments, the light activated protein comprises an N-terminal signalpeptide, a C-terminal ER Export signal, and a C-terminal traffickingsignal. In some embodiments, the light activated protein comprises aC-terminal ER Export signal and a C-terminal trafficking signal. In someembodiments, the C-terminal ER Export signal and the C-terminaltrafficking signal are linked by a linker. The linker can comprise anyof 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275,300, 400, or 500 amino acids in length. The linker may further comprisea fluorescent protein, for example, but not limited to, a yellowfluorescent protein, a red fluorescent protein, a green fluorescentprotein, or a cyan fluorescent protein. In some embodiments the ERExport signal is more C-terminally located than the trafficking signal.In some embodiments the trafficking signal is more C-terminally locatedthan the ER Export signal.

Also provided herein are isolated polynucleotides encoding any of theproteins described herein, such as a light-activated protein comprisinga core amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 1.Also provided herein are expression vectors (such as a viral vectordescribed herein) comprising a polynucleotide encoding any of theproteins described herein, such as a light-activated protein comprisinga core amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 1.The polynucleotides may be used for expression of the light-activatedprotein in animal cells.

Provided herein is an animal cell comprising a light-activated proteinexpressed on the cell membrane, wherein the protein is responsive toblue light and is derived from Dunaliella salina, wherein the protein iscapable of mediating a depolarizing current in the cell when the cell isilluminated with light. In some embodiments the light has a wavelengthbetween about 450 and 495 nm. In some embodiments, the light has awavelength about 490 nm. In some embodiments, the light-activatedprotein comprises an amino acid sequence at least 95% identical to thesequence shown in residues 25-365, 24-365, 23-365, 22-365, 21-365,20-365, 19-365, 18-365, 17-365, 16-365, 15-365, 14-365, 13-365, 13-365,12-365, 11-365, 10-365, 9-365, 8-365, 7-365, 6-365, 5-365, 4-365, 3-365,2-365, or 1-365 of SEQ ID NO: 2. In some embodiments, thelight-activated protein comprises an amino acid sequence at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence shown in residues 25-365, 24-365, 23-365, 22-365, 21-365,20-365, 19-365, 18-365, 17-365, 16-365, 15-365, 14-365, 13-365, 13-365,12-365, 11-365, 10-365, 9-365, 8-365, 7-365, 6-365, 5-365, 4-365, 3-365,2-365, or 1-365 of SEQ ID NO: 2. In some embodiments, thelight-activated protein comprises substitutions, deletions, and/orinsertions introduced into a native amino acid sequence to increase ordecrease sensitivity to light, increase or decrease sensitivity toparticular wavelengths of light, and/or increase or decrease the abilityof the light-activated protein to regulate the polarization state of theplasma membrane of the cell. In some embodiments, the light-activatedprotein contains one or more conservative amino acid substitutions. Insome embodiments, the light-activated protein contains one or morenon-conservative amino acid substitutions. The light-activated proteincomprising substitutions, deletions, and/or insertions introduced intothe native amino acid sequence suitably retains the ability todepolarize the plasma membrane of a neuronal cell in response to light.

Also provided herein is an animal cell comprising a light-activatedprotein expressed on the cell membrane, wherein the protein comprises acore amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in residues25-365, 24-365, 23-365, 22-365, 21-365, 20-365, 19-365, 18-365, 17-365,16-365, 15-365, 14-365, 13-365, 13-365, 12-365, 11-365, 10-365, 9-365,8-365, 7-365, 6-365, 5-365, 4-365, 3-365, 2-365, or 1-365 of SEQ ID NO:2 and an endoplasmic reticulum (ER) export signal. The ER export signalmay be fused to the C-terminus of the core amino acid sequence or may befused to the N-terminus of the core amino acid sequence. In someembodiments, the ER export signal is linked to the core amino acidsequence by a linker. The linker can comprise any of 5, 10, 20, 30, 40,50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 aminoacids in length. The linker may further comprise a fluorescent protein,for example, but not limited to, a yellow fluorescent protein, a redfluorescent protein, a green fluorescent protein, or a cyan fluorescentprotein. In some embodiments, the ER export signal comprises the aminoacid sequence FXYENE, where X can be any amino acid. In someembodiments, the ER export signal comprises the amino acid sequenceVXXSL, where X can be any amino acid. In some embodiments, the ER exportsignal comprises the amino acid sequence FCYENEV.

Also provided herein is an animal cell comprising a light-activatedprotein expressed on the cell membrane, wherein the protein comprises acore amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in residues25-365, 24-365, 23-365, 22-365, 21-365, 20-365, 19-365, 18-365, 17-365,16-365, 15-365, 14-365, 13-365, 13-365, 12-365, 11-365, 10-365, 9-365,8-365, 7-365, 6-365, 5-365, 4-365, 3-365, 2-365, or 1-365 of SEQ ID NO:2 and a signal peptide (e.g., which enhances transport to the plasmamembrane). The signal peptide may be fused to the C-terminus of the coreamino acid sequence or may be fused to the N-terminus of the core aminoacid sequence. In some embodiments, the signal peptide is linked to thecore amino acid sequence by a linker. The linker can comprise any of 5,10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,400, or 500 amino acids in length. The linker may further comprise afluorescent protein, for example, but not limited to, a yellowfluorescent protein, a red fluorescent protein, a green fluorescentprotein, or a cyan fluorescent protein. In some embodiments, the signalpeptide comprises the amino acid sequenceMDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNG. In some embodiments,other signal peptides (such as signal peptides from other opsins) may beused.

Also provided herein is an animal cell comprising a light-activatedprotein expressed on the cell membrane, wherein the protein comprises acore amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in residues25-365, 24-365, 23-365, 22-365, 21-365, 20-365, 19-365, 18-365, 17-365,16-365, 15-365, 14-365, 13-365, 13-365, 12-365, 11-365, 10-365, 9-365,8-365, 7-365, 6-365, 5-365, 4-365, 3-365, 2-365, or 1-365 of SEQ ID NO:2 and a trafficking signal (e.g., which enhances transport to the plasmamembrane). The signal peptide may be fused to the C-terminus of the coreamino acid sequence or may be fused to the N-terminus of the core aminoacid sequence. In some embodiments, the signal peptide is linked to thecore amino acid sequence by a linker. The linker can comprise any of 5,10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,400, or 500 amino acids in length. The linker may further comprise afluorescent protein, for example, but not limited to, a yellowfluorescent protein, a red fluorescent protein, a green fluorescentprotein, or a cyan fluorescent protein. In some embodiments, thetrafficking signal is derived from the amino acid sequence of the humaninward rectifier potassium channel Kir2.1. In some embodiments, thetrafficking signal comprises the amino acid sequenceKSRITSEGEYIPLDQIDINV.

Also provided herein is an animal cell comprising a light-activatedprotein expressed on the cell membrane, wherein the protein comprises acore amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in residues25-365, 24-365, 23-365, 22-365, 21-365, 20-365, 19-365, 18-365, 17-365,16-365, 15-365, 14-365, 13-365, 13-365, 12-365, 11-365, 10-365, 9-365,8-365, 7-365, 6-365, 5-365, 4-365, 3-365, 2-365, or 1-365 of SEQ ID NO:2 and two or more amino acid sequence motifs which enhance transport tothe plasma membranes of mammalian cells selected from the groupconsisting of a signal peptide, an ER export signal, and a membranetrafficking signal. In some embodiments, the light activated proteincomprises an N-terminal signal peptide and a C-terminal ER exportsignal. In some embodiments, the light activated protein comprises anN-terminal signal peptide and a C-terminal trafficking signal. In someembodiments, the light activated protein comprises an N-terminal signalpeptide, a C-terminal ER Export signal, and a C-terminal traffickingsignal. In some embodiments, the light activated protein comprises aC-terminal ER Export signal and a C-terminal trafficking signal. In someembodiments, the C-terminal ER Export signal and the C-terminaltrafficking signal are linked by a linker. The linker can comprise anyof 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275,300, 400, or 500 amino acids in length. The linker may further comprisea fluorescent protein, for example, but not limited to, a yellowfluorescent protein, a red fluorescent protein, a green fluorescentprotein, or a cyan fluorescent protein. In some embodiments the ERExport signal is more C-terminally located than the trafficking signal.In some embodiments the trafficking signal is more C-terminally locatedthan the ER Export signal.

Also provided herein are isolated polynucleotides encoding any of theproteins described herein, such as a light-activated protein comprisinga core amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in residues25-365, 24-365, 23-365, 22-365, 21-365, 20-365, 19-365, 18-365, 17-365,16-365, 15-365, 14-365, 13-365, 13-365, 12-365, 11-365, 10-365, 9-365,8-365, 7-365, 6-365, 5-365, 4-365, 3-365, 2-365, or 1-365 of SEQ ID NO:2. Also provided herein are expression vectors (such as a viral vectordescribed herein) comprising a polynucleotide encoding any of theproteins described herein, such as a light-activated protein comprisinga core amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in residues25-365, 24-365, 23-365, 22-365, 21-365, 20-365, 19-365, 18-365, 17-365,16-365, 15-365, 14-365, 13-365, 13-365, 12-365, 11-365, 10-365, 9-365,8-365, 7-365, 6-365, 5-365, 4-365, 3-365, 2-365, or 1-365 of SEQ ID NO:2. The polynucleotides may be used for expression of the light-activatedprotein in animal cells.

Provided herein is an animal cell comprising a light-activated proteinexpressed on the cell membrane, wherein the protein is responsive toamber as well as red light and is derived from Natronomonas pharaonic,wherein the protein is capable of mediating a hyperpolarizing current inthe cell when the cell is illuminated with light. In some embodimentsthe light has a wavelength between about 580 and 630 nm. In someembodiments, the light has a wavelength about 589 nm. In someembodiments, the light has a wavelength greater than about 630 nm (e.g.less than 740 nm). In some embodiments, the light has a wavelengtharound 630 nm. In some embodiments, the light-activated proteincomprises an amino acid sequence at least 95% identical to the sequenceshown in SEQ ID NO: 3. In some embodiments, the light-activated proteincomprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ IDNO: 3. In some embodiments, the light-activated protein comprisessubstitutions, deletions, and/or insertions introduced into a nativeamino acid sequence to increase or decrease sensitivity to light,increase or decrease sensitivity to particular wavelengths of light,and/or increase or decrease the ability of the light-activated proteinto regulate the polarization state of the plasma membrane of the cell.In some embodiments, the light-activated protein contains one or moreconservative amino acid substitutions. In some embodiments, thelight-activated protein contains one or more non-conservative amino acidsubstitutions. The light-activated protein comprising substitutions,deletions, and/or insertions introduced into the native amino acidsequence suitably retains the ability to hyperpolarize the plasmamembrane of a neuronal cell in response to light.

Also provided herein is an animal cell comprising a light-activatedprotein expressed on the cell membrane, wherein the protein comprises acore amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 3and an endoplasmic reticulum (ER) export signal. The ER export signalmay be fused to the C-terminus of the core amino acid sequence or may befused to the N-terminus of the core amino acid sequence. In someembodiments, the ER export signal is linked to the core amino acidsequence by a linker. The linker can comprise any of 5, 10, 20, 30, 40,50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 aminoacids in length. The linker may further comprise a fluorescent protein,for example, but not limited to, a yellow fluorescent protein, a redfluorescent protein, a green fluorescent protein, or a cyan fluorescentprotein. In some embodiments, the ER export signal comprises the aminoacid sequence FXYENE, where X can be any amino acid. In someembodiments, the ER export signal comprises the amino acid sequenceVXXSL, where X can be any amino acid. In some embodiments, the ER exportsignal comprises the amino acid sequence FCYENEV.

Also provided herein is an animal cell comprising a light-activatedprotein expressed on the cell membrane, wherein the protein comprises acore amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 3and a trafficking signal (e.g., which enhances transport to the plasmamembrane). The signal peptide may be fused to the C-terminus of the coreamino acid sequence or may be fused to the N-terminus of the core aminoacid sequence. In some embodiments, the signal peptide is linked to thecore amino acid sequence by a linker. The linker can comprise any of 5,10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,400, or 500 amino acids in length. The linker may further comprise afluorescent protein, for example, but not limited to, a yellowfluorescent protein, a red fluorescent protein, a green fluorescentprotein, or a cyan fluorescent protein. In some embodiments, thetrafficking signal is derived from the amino acid sequence of the humaninward rectifier potassium channel Kir2.1. In some embodiments, thetrafficking signal comprises the amino acid sequence K S R I T S E G E YI P L D Q I D I N V.

Also provided herein is an animal cell comprising a light-activatedprotein expressed on the cell membrane, wherein the protein comprises acore amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 3and two or more amino acid sequence motifs which enhance transport tothe plasma membranes of mammalian cells selected from the groupconsisting of an ER export signal and a membrane trafficking signal. Insome embodiments, the light activated protein comprises a C-terminal ERExport signal and a C-terminal trafficking signal. In some embodiments,the C-terminal ER Export signal and the C-terminal trafficking signalare linked by a linker. The linker can comprise any of 5, 10, 20, 30,40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500amino acids in length. The linker may further comprise a fluorescentprotein, for example, but not limited to, a yellow fluorescent protein,a red fluorescent protein, a green fluorescent protein, or a cyanfluorescent protein. In some embodiments the ER Export signal is moreC-terminally located than the trafficking signal. In some embodimentsthe trafficking signal is more C-terminally located than the ER Exportsignal.

Also provided herein is an animal cell comprising a light-activatedprotein expressed on the cell membrane, wherein the protein comprises acore amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 3,wherein the N-terminal signal peptide of SEQ ID NO:3 is deleted orsubstituted In some embodiments, the light-activated protein comprisesan amino acid sequence at least 95% identical to the sequence shown inresidues 40-291, 39-291, 38-291, 37-291, 36-291, 35-291, 34-291, 33-291,32-291, 31-291, 30-291, 29-291, 28-291, 27-291, 26-291, 25-291, 24-291,23-291, 22-291, 21-291, 20-291, 19-291, 18-291, 17-291, 16-291, 15-291,14-291, 13-291, 13-291, 12-291, 11-291, 10-291, 9-291, 8-291, 7-291,6-291, 5-291, 4-291, 3-291, 2-291, or 1-291 of SEQ ID NO:3. In someembodiments, other signal peptides (such as signal peptides from otheropsins) may be used. The light-activated protein may further comprise anER transport signal and a membrane trafficking signal described herein.

Also provided herein are polynucleotides encoding for any of theproteins described herein, such as a light-activated protein comprisinga core amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 3,an ER export signal, and a membrane trafficking signal. Thepolynucleotides may be in an expression vector (such as a viral vectordescribed herein). The polynucleotides may be used for expression of thelight-activated protein in animal cells.

Provided herein is an animal cell comprising a light-activated proteinexpressed on the cell membrane, wherein the protein is responsive togreen light and is derived from Halobacterium salinarum wherein theprotein is capable of mediating a hyperpolarizing current in the cellwhen the cell is illuminated with light. In some embodiments the lighthas a wavelength between about 520 and 570 nm. In some embodiments, thelight has a wavelength about 560 nm. In some embodiments, thelight-activated protein comprises an amino acid sequence at least 95%identical to the sequence shown in SEQ ID NO: 4. In some embodiments,the light-activated protein comprises an amino acid sequence at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical tothe sequence shown in SEQ ID NO: 4. In some embodiments, thelight-activated protein comprises substitutions, deletions, and/orinsertions introduced into a native amino acid sequence to increase ordecrease sensitivity to light, increase or decrease sensitivity toparticular wavelengths of light, and/or increase or decrease the abilityof the light-activated protein to regulate the polarization state of theplasma membrane of the cell. In some embodiments, the light-activatedprotein contains one or more conservative amino acid substitutions. Insome embodiments, the light-activated protein contains one or morenon-conservative amino acid substitutions. The light-activated proteincomprising substitutions, deletions, and/or insertions introduced intothe native amino acid sequence suitably retains the ability tohyperpolarize the plasma membrane of a neuronal cell in response tolight.

Also provided herein is an animal cell comprising a light-activatedprotein expressed on the cell membrane, wherein the protein comprises acore amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 4and an endoplasmic reticulum (ER) export signal. The ER export signalmay be fused to the C-terminus of the core amino acid sequence or may befused to the N-terminus of the core amino acid sequence. In someembodiments, the ER export signal is linked to the core amino acidsequence by a linker. The linker can comprise any of 5, 10, 20, 30, 40,50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 aminoacids in length. The linker may further comprise a fluorescent protein,for example, but not limited to, a yellow fluorescent protein, a redfluorescent protein, a green fluorescent protein, or a cyan fluorescentprotein. In some embodiments, the ER export signal comprises the aminoacid sequence FXYENE, where X can be any amino acid. In someembodiments, the ER export signal comprises the amino acid sequenceVXXSL, where X can be any amino acid. In some embodiments, the ER exportsignal comprises the amino acid sequence FCYENEV.

Also provided herein is an animal cell comprising a light-activatedprotein expressed on the cell membrane, wherein the protein comprises acore amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 4and a trafficking signal (e.g., which enhances transport to the plasmamembrane). The signal peptide may be fused to the C-terminus of the coreamino acid sequence or may be fused to the N-terminus of the core aminoacid sequence. In some embodiments, the signal peptide is linked to thecore amino acid sequence by a linker. The linker can comprise any of 5,10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,400, or 500 amino acids in length. The linker may further comprise afluorescent protein, for example, but not limited to, a yellowfluorescent protein, a red fluorescent protein, a green fluorescentprotein, or a cyan fluorescent protein. In some embodiments, thetrafficking signal is derived from the amino acid sequence of the humaninward rectifier potassium channel Kir2.1. In some embodiments, thetrafficking signal comprises the amino acid sequence K S R I T S E G E YI P L D Q I D I N V.

Also provided herein is an animal cell comprising a light-activatedprotein expressed on the cell membrane, wherein the protein comprises acore amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 4and two or more amino acid sequence motifs which enhance transport tothe plasma membranes of mammalian cells selected from the groupconsisting of an ER export signal and a membrane trafficking signal. Insome embodiments, the light activated protein comprises a C-terminal ERExport signal and a C-terminal trafficking signal. In some embodiments,the C-terminal ER Export signal and the C-terminal trafficking signalare linked by a linker. The linker can comprise any of 5, 10, 20, 30,40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500amino acids in length. The linker may further comprise a fluorescentprotein, for example, but not limited to, a yellow fluorescent protein,a red fluorescent protein, a green fluorescent protein, or a cyanfluorescent protein. In some embodiments the ER Export signal is moreC-terminally located than the trafficking signal. In some embodimentsthe trafficking signal is more C-terminally located than the ER Exportsignal.

Also provided herein is an animal cell comprising a light-activatedprotein expressed on the cell membrane, wherein the protein comprises acore amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 4,wherein the N-terminal signal peptide of SEQ ID NO:4 is deleted orsubstituted. In some embodiments, the light-activated protein comprisesan amino acid sequence at least 95% identical to the sequence shown inresidues 40-262, 39-262, 38-262, 37-262, 36-262, 35-262, 34-262, 33-262,32-262, 31-262, 30-262, 29-262, 28-262, 27-262, 26-262, 25-262, 24-262,23-262, 22-262, 21-262, 20-262, 19-262, 18-262, 17-262, 16-262, 15-262,14-262, 13-262, 13-262, 12-262, 11-262, 10-262, 9-262, 8-262, 7-262,6-262, 5-262, 4-262, 3-262, 2-262, or 1-262 of SEQ ID NO:4. In someembodiments, other signal peptides (such as signal peptides from otheropsins) may be used. The light-activated protein may further comprise anER transport signal and/or a membrane trafficking signal describedherein.

Also provided herein are polynucleotides encoding for any of theproteins described herein, such as a light-activated protein comprisinga core amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 4,an ER export signal, and a membrane trafficking signal. Thepolynucleotides may be in an expression vector (such as a viral vectordescribed herein). The polynucleotides may be used for expression of thelight-activated protein in animal cells.

In certain more particular embodiments, a method for providing a lightresponsive protein for mammalian expression is provided. A lightresponsive protein is isolated from G. theta (GtR3). GtR3 has aC-terminus and an N-terminus. A promoter is added to the C-terminus ofGtR3. In certain embodiments the promoter is an endoplasmic reticulum(ER) export signal. For more specifics on optimizing GtR3 for expressionin mammalian cells, see Appendix A as filed in the underlyingprovisional application and entitled, “Molecular and Cellular Approachesfor Diversifying and Extending Optogenetics.”

An excitatory protein encourages firing of the action potential bymoving the potential of the cell toward the action potential triggerlevel for the cell. In many neurons, this means that the proteindecreases the negative voltage seen across the cell membrane. In aspecific instance, the protein acts an additional ion channel thattransfers cations into the cell. In this manner, the protein generatesan excitatory current across the cell membrane. More specifically, theprotein responds to light by raising the voltage across the cellmembrane, thereby increasing the probability that an action potential ordepolarization will occur. In certain instances the voltage across thecell membrane can be increased to the action potential trigger level orhigher, causing depolarization of the cell.

Certain aspects of the present invention are based on the identificationand development of a channel (DChR) that is derived from DunaliellaChannelrhodopsin DChR mediates a robust depolarizing current whenilluminated with blue light. The depolarizing current may cause a cellto become excited in response to exposure to blue light. In certainembodiments DChR is expressed as an exogenous protein in a cell ofinterest. For neural cells, DChR can function as an excitatory proteinthat uses an endogenous cofactor when delivered to a cell of interest.

The introduction of GtR3 and DChR to the field of optogenetics opens thedoor for a variety of applications. For instance, GtR3 and DChR can beused in combination with other optogenetic ion-passing molecules, suchas ChR2 (developed from Chlamydomonas reinhardtii). GtR3 and DChR havedifferent operating parameters, relative to other light-responsivemolecules. This facilitates the development of a cell or a cellpopulation that has a precisely-tuned response to different wavelengthsof light. The parameter that can be tuned include, but are not limitedto, current density, hyperpolarization level, membrane conductance,stimulus frequency, resting potential, optical wavelength and/or opticalintensity.

In another example embodiment, a method for controlling action potentialof a neuron involves the following steps: engineering a first lightresponsive protein in the neuron; producing, in response to light, aninhibitory current in the neuron and from the first light responsiveprotein; engineering a second light responsive protein in the neuron;and producing, in response to light, an excitation current in the neuronfrom the second light responsive protein. The first light responsiveprotein and the second light responsive protein may be responsive todifferent wavelengths of light. In certain instances, the use of DChR orGtR3 can facilitate specific current densities for this excitation orinhibitory current and/or optical wavelength at which such current isgenerated.

In another example embodiment, a method for controlling action potentialof a neuron involves the following steps: engineering a first lightresponsive protein in the neuron; producing, in response to light, aninhibitory current in the neuron and from the first light responsiveprotein; engineering a second light responsive protein in the neuron;and producing, in response to light, a second inhibitory current fromthe second light responsive protein, the combination of the two currentsmore strongly inhibiting the neuron than a single light responsiveprotein. For instance, the first light responsive protein could be NpHRand the second light responsive protein could be GtR3.

Another method for controlling a voltage level across a cell membrane ofa cell includes measuring the voltage level of the cell membrane. Alight responsive protein is engineered and introducing the lightresponsive protein into the cell, where it is expressed. A light of aparticular wavelength is provided, which produces a reaction in the cellby the light responsive protein. The response by the light responsiveprotein produces a current across the cell membrane. The current isresponsive to the measured voltage level.

Another aspect of the present invention is directed to a system forcontrolling an action potential of a neuron in vivo. The system includesa delivery device, a light source, and a control device. The deliverydevice introduces a light responsive protein to the neuron, with thelight responsive protein producing an inhibitory current. The lightsource generates light for stimulating the light responsive protein, andthe control device controls the generation of light by the light source.In other embodiments the light responsive protein introduced into theneuron produces an excitatory current.

In more detailed embodiments, such a system is further adapted such thatthe delivery device introduces the light responsive protein by one oftransfection, transduction and microinjection, and/or such that thelight source introduces light to the neuron via one of an implantablelight generator and fiber-optics. For additional detail regarding methodof delivery and expression of genes, see, PCT Publication No.WO2010/019619 A1 (PCT/US2009/053474), entitled “Method and Compositionfor Controlling Gene Expression,” which is fully incorporated herein byreference.

Another aspect of the present disclosure is directed to a method fortreatment of a disorder. The method targets a group of neuronsassociated with the disorder; and in this group, the method includesengineering inhibitory proteins to respond to light by producing aninhibitory current to dissuade depolarization of the neurons, andexposing the neurons to light, thereby dissuading depolarization of theneurons.

Yet another aspect of the present disclosure is directed to a furthermethod for treatment of a disorder. The method targets a group ofneurons associated with the disorder; and in this group, the methodincludes engineering excitatory proteins to respond to light byproducing an excitatory current to encourage depolarization of theneurons, and exposing the neurons to light, thereby encouragingdepolarization of the neurons. In other aspects of the present inventionboth inhibitory and excitatory proteins, responsive to differentwavelengths of light, are introduced into a targeted group of neurons.The neurons are exposed alternatively to different wavelengths of lightto excite, and inhibit the depolarization of the neurons.

More detailed embodiments expand on such techniques. For instance,another aspect of the present invention co-expresses GtR3 and DChR withother known opsins, such as NpHR and VChR1 in the species (e.g., a mouseand C. elegans). Also, opsins creating currents of opposite polarity andhaving different color sensitivity are integrated with calcium imagingin acute mammalian brain slices for bidirectional optical modulation andreadout of neural activity. Likewise, the coupled opsins can be targetedto C. elegans muscle and cholinergic motoneurons to control locomotionbidirectionally. Together coupled opsins can be used as a complete andcomplementary optogenetic system for multimodal, high-speed,genetically-targeted, all-optical interrogation of living neuralcircuits. Further, more than one set of cells may be targeted withdifferent couplings of opsins, allowing for precise control of multiplesets of cells independently from each other.

In addition to GtR3 and DChR, there are a number of channelrhodopsins,halorhodopsins, and microbial opsins that can be engineered to opticallyregulate ion flux or second messengers within cells. Various embodimentsof the invention include codon-optimized, mutated, truncated, fusionproteins, targeted versions, or otherwise modified versions of such ionoptical regulators. For example, GtR3 and DChR (e.g., Appendices B and Cas filed in the underlying provisional application). The cited opsinsare used as representative of a number of different embodiments.Discussions specifically identifying GtR3 and DChR are not meant tolimit the invention to such specific examples of optical regulatorsunless specified. For further details regarding the above mentionedsequences, reference can be made to “Multimodal fast opticalinterrogation of neural circuitry” by Feng Zhang, et al, Nature (Apr. 5,2007) Vol. 446: 633-639, which is fully incorporated herein byreference.

Table 1 shows identified opsins for inhibition of cellular activityacross the visible spectrum:

TABLE 1 Fast optogenetics: inibition across the visible spectrumBiological Wavelength Opsin Type Origin Sensitivity Defined action NpHRnatronomonas 680 nm utility Inhibition pharaonis (with 3.0 series)(hyperpolarization) 589 nm max BR halobacterium 570 nm max Inhibitionhelobium (hyperpolarization) AR Acetabulaira 518 nm max Inhibitionacetabulum (hyperpolarization) GtR3 Guillardia 472 nm max Inhibitiontheta (hyperpolarization)Table 2 shows identified opsins for excitation and modulation across thevisible spectrum:

TABLE 2 Fast optogenetics: excitation and modulation across the visiblespectrum Opsin Wavelength Type Biological Origin Sensitivity Definedaction VChR1 Volvox carteri 589 nm utility Excitation 535 nm max(depolarization) optoXRs Bos taurus 505 nm max Modulation (Gs, Gqpathways) DChR Dunaliella salina 500 nm max Excitation (depolarization)SFOs Chlamydomonas 546 nm deactivate modulation reinhardtii 470 nmactivate (electrical up states) ChR2 Chlamydomonas 470 nm max Excitationreinhardtii 380-405 nm utility (depolarization)

Opsins described in U.S. patent application Ser. Nos. 12/988,567 and12/996,753, U.S. Patent Application Publication Nos: 2007/0054319,2010/0234273, 2007/0261127, 2007/0053996, 2010/0145418, 2009/0093403,2008/0085265, 2010/0190229, 2009/0099038, and PCT Publication No.PCT/US09/64355 are incorporated herein by reference in their entirety.

In certain embodiments of the present invention, various combinations ofthe listed opsins, for example, are delivered to a cell population. Asecond combination can be delivered to a different cell population.Using the defined action and the wavelength sensitivity information foreach opsin, combinations of opsins may be chosen to excite certain cellpopulations while inhibiting others, for example.

Consistent with one example embodiment of the present invention, targetcells are stimulated using an implantable arrangement. The implantablearrangement includes a biological portion that facilitates thestimulation of the target cells in response to receipt of light. Theimplantable arrangement also includes a light generator for creatinglight to trigger the stimulus of the target cells. In certainembodiments the implantable arrangement includes a microcontroller and afeedback loop. The color of light created by the light generator isdependent on the voltage of the cell. The target cell can includemultiple light sensitive proteins which respond to different wavelengthsof light to inhibit or encourage depolarization of the target cell.

Consistent with another example embodiment of the present invention, amethod is implemented for stimulating target cells in vivo using genetransfer vectors (for example, viruses) capable of inducingphotosensitive ion channel growth (for example, DChR ion channels) orphotosensitive ion pumps (for example, GtR3 proton pumps). The vectorsare implanted in the body, along with the electronic components of theapparatus. A light producing device is implanted near the target cells.The target cells are stimulated in response to light generated by thelight producing device. In certain more specific embodiments, genetransfer vectors are capable of inducing growth of more than one ionchannel or ion pump.

Consistent with a particular embodiment of the present invention, aprotein is introduced to one or more target cells. When introduced intoa cell, the protein changes the potential of the cell in response tolight having a certain frequency. This may result in a change in restingpotential that can be used to control (dissuade) action potentialfiring. In a specific example, the protein is GtR3 which acts as amembrane pump for transferring charge across the cell membrane inresponse to light. Membrane pumps are energy transducers which useelectromagnetic or chemical bond energy for translocation of ions acrossthe membrane. In other specific examples, the protein is a halorhodopsinwhich acts as the membrane pump. The halorhodopsin membrane pump movesspecific ions across the membrane. For further information regardinghalorhodopsin membrane pumps, reference can be made to “Halorhodopsin Isa Light-driven Chloride Pump” by Brigitte Schobert et al, The Journal ofBiological Chemistry Vol. 257, No. 17. Sep. 10, 1982, pp. 10306-10313,which is fully incorporated herein by reference.

In specific embodiments GtR3 and NpHR may be introduced into differenttarget cell populations, respectively. The two inhibitors are responsiveto different wavelengths of light allowing the two proteins to be usedto inhibit different target cell populations independently. GtR3 andNpHR may also be introduced into the same cell population for gradientcontrol over the inhibition of a cell population. For instance, GtR3 andNpHR molecules within the same cell can be activated relativelyindependent from one another. Thus, a first level of inhibition can beimplemented by activating the molecule type (GtR3 or NpHR) that providesthe lowest current levels for the cell conditions. The next level couldbe implemented by deactivating this first molecule type and activatingthe other molecule type. A third level can be implemented by activatingboth molecules simultaneously. Further gradient control can be achievedby varying the wavelength of light, or by varying the light intensity ator near the maxima of each protein, for example.

The combination of multiple inhibitors can also lead to shuntinginhibition, which combined with hyperpolarizing inhibition can lead tostronger hyperpolarization in the combination approach than from using asingle inhibitor protein. In a cell membrane the reversal potential(also known as the Nernst potential) of an ion is the membrane potentialat which there is no net (overall) flow of ions from one side of themembrane to the other. Each type of ion channel has a specific reversalpotential. At the Nernst potential the outward and inward rates of ionmovement are the same (the ion flux is in equilibrium for the ion type).A change of membrane potential on either side of the Nernst potentialreverses the overall direction of ion flux. If the reversal potential ofan ion channel is below the resting potential (or instant potential inthe case where a light responsive protein ion channel been stimulatedand changed the “resting” potential) of the cell membrane, the ionchannel will contribute to inhibition through hyperpolarization of thecell. In contract, if the reversal potential for an ion channel isbetween the resting potential and the threshold for the generation ofaction potentials, the ion channel will have a shunting effect. Theconcentration gradient for any given ion (for example Cl—) is determinedin part by the balance between the activity of the other ions (forexample Na+ and K+). Shunting inhibition is termed “shunting” becausethe channel conductance short-circuits currents that are generated atadjacent excitatory channels. Accordingly, the addition of a lightresponsive protein channel with inhibitory characteristics can lower themembrane potential to a point where an existing ion channel reversesdirection and “shunts” the current, creating an additional path whichprevents the membrane potential from reaching the action potential.

Provided herein are populations of cells, tissues, and non-human animalscomprising a cell expressing one or more of the light-activated proteinsdescribed herein on the cell membrane. Methods of using the cells,population of cells, tissues, and non-human animals are also provided.

FIG. 1A shows a light responsive ion-passing molecule (a pump orchannel) 150 in a cell membrane 160. In general, ion channels allow flowtoward the channel's reversal potential (or equilibrium potential forthe channel); the direction is based on whether the membrane potentialis above or below the channel's reversal potential. Ion channels do notrequire additional energy to transport the ion. Ion pumps transport ionsagainst the equilibrium flow and require introduction of some form ofenergy, such as ATP, to transport the ion across the membrane. Ions 162may be anions or cations. The hydrogen ion is a proton (as well as acation). The direction the ions flow along with the ions polarity willdetermine whether the ion-passing molecule has a hyperpolarizing effector a depolarizing effect on the cell. In certain embodiments, theion-passing molecule 150 transports protons out of the cell, causinghyperpolarization of the cell and inhibition of the firing of the cell.The light responsive ion channel/pump 150 is not open/active unlesslight of the appropriate frequency is present.

FIG. 1B shows an alternative embodiment of the present invention where alight responsive ion-passing molecule 150 is expressed within membranesthat are internal to a cell. Many of the internal components of the cellhave membranes similar to the cell membrane that encloses the cell. Themembrane of organelle 152 contains naturally occurring ion pump/channelsbefore introduction of a light responsive protein. Depending on thepromoters introduced along with the exogenous light responsive protein,the light responsive protein can express as an ion pump/channel in themembrane of variety of different organelles, in the cell membrane, or acombination of different organelles and/or the cell membrane. In certainembodiments the light responsive ion-passing molecule 150 is a protonpump. In other embodiments the pump can be a pump/channel for a specificcation or a specific anion. The ion pump/channel 150 is located in themembrane 156 of the organelle.

The transfer of protons across a membrane in which the ion-passingmolecule is expressed can be used to change the membrane potential ofeither or both membrane 156 and membrane 160. The transport of protonsout of the cytoplasm can cause a cell to become hyperpolarized,resulting in inhibition of the cell.

In certain instances, the pump/channel 150 can be used to change the pHof the organelle 152 and the surrounding cytoplasm 154. In addition, thepH of the organelle in which the protein is expressed can be changedand/or controlled. Because most enzymes within a cell are pH sensitive,the pH of each organelle critically determines the coordinatedbio-chemical reactions occurring along the endocytic and secretorypathways. Aberrations of the normal organellar pH homeostasis can leadto significant functional changes. Depending on the level of expressionof the light responsive protein, and the location (i.e. which organelle)various functions of the cell may be enhanced or retarded. At highenough levels of expression the cell may be killed. This can bedesirable in unwanted (i.e. cancer) cells. For instance, cells can belocally transfected at a cancerous tumor location and then carefullydirected light pulses can be used to shrink or eliminate the tumor.

FIG. 2A shows two examples of the use of light responsive proteins, eachexample including three cell populations, A, B, and C. As discussedbriefly above, two or more light responsive proteins can be introducedinto the same cell populations. The light responsive proteins introducedcan cause the same response or different response when activated. Thedifferent proteins can also have different reaction times, or strengthsof reactions. Two proteins of the same polarity can be activated at thesame time causing an additive effect. Two depolarizing proteins, forexample, may be alternatively stimulated. This can allow for an increasein frequency of firing. In example 1, cell populations A and B expressthe same inhibiting protein, but different excitatory proteins. Cellpopulation C has an excitatory protein which reacts to light around thesame wavelength as the inhibiting protein of cell populations A and B.The inhibitor of cell population C reacts to a wavelength similar to theexcitatory protein of cell population A. This arrangement allows for avariety of combinations of cell population reactions to the lightprovided to stimulate the cell populations. As example 1 is set up, cellpopulation B may be excited without exciting or inhibiting either of theother two populations. However, cell populations A and C react in almostan inverse way to the light provided.

Example 2 includes cell population A with three light responsiveproteins and cell populations B and C with two light responsiveproteins. Cell population A includes two excitatory proteins which reactto different light wavelengths, and one inhibitory protein. Cellpopulations B and C each have one of the excitatory proteins of cellpopulation A and an inhibitory protein which responses to wavelengths inthe same spectrum as the excitatory protein present in the other cellpopulation (B or C). This arrangement allows for the combination of cellpopulations A and B to be excited, or cell populations B and C to beexcited, while the third cell population is inhibited. It allows forcell population A to be inhibited without affecting either cellpopulation B or C, and for cell population A to be excited while bothcell populations B and C are inhibited. The example combination of FIG.2A are not meant to be limiting, but to illustrate some of the manycombinations of light responsive proteins in both a single cellpopulation, and the combinations across cell populations.

Consistent with another example embodiment of the present invention,target cells are neurons located in the brain of a mammal. The targetcells are genetically modified to express photosensitive bio-moleculararrangement, for example, DChR ion channels. Light can then be used tostimulate the neurons. Depending upon a number of factors, such as thelocation within the brain and the frequency and length of stimulation,different objectives can be achieved. For instance, current techniquesfor deep brain stimulus (DBS) use electrodes to apply a current directlyto the targeted area of the brain. The frequency of the electricalstimulus is sometimes referred to as either low-frequency DBS orhigh-frequency DBS. Studies have suggested that high-frequency DBSinhibits the generation of impulses from the stimulated cells, whilelow-frequency DBS facilitates the generation of impulses from thestimulated cells. The frequencies that produce the effects ofhigh-frequency of low-frequency DBS have also been shown to varydepending upon the specific area of the brain being stimulated.According to one example of high-frequency DBS, the neurons arestimulated using electrodes supplying current pulses at frequenciesaround 100 Hz or more. Such a frequency has been shown to be effectivein certain applications, as discussed further herein.

In various cell populations, similar to those illustrated in FIG. 2A,two or more light responsive proteins may be present. Some (or all) ofthe two or more light responsive proteins can direct the same action(i.e., excitation or inhibition) within the cell population. For examplecell population A of example 2 includes two excitatory light responsiveproteins, DChR and VChR1. FIG. 2B shows a stimulus profile for use withcertain embodiments in which two or more types of light responsiveproteins, for example DChR and VChR1, may be introduced into the samecell population. Each ion-passing molecule has a respective stimulusfrequency limit, which can be partially responsive to environmentalfactors like pH. This limit reflects the recovery time necessary for themolecule to transition between active and inactive states. The use oftwo types of light-responsive proteins, each responding to differentoptical wavelengths, can allow for each type of protein to be controlledseparately. The respective ion channels can be activated alternatively,allowing for an increased frequency of stimulation of the brain.

For instance, example 3 shows that light pulses (solid vertical lines)of a first wavelength and provided at a frequency F. This wavelength isselected to activate a first type of molecule (triangles). A second setof light pulses (dotted vertical lines) of a second wavelength can alsobe provided at a frequency F. This second set of light pulses can beused to activate a second type of molecule (squares). The resultingactivation frequency for both cells is thus twice the frequency ofactivation for each individual ion-passing molecule. Of course, thefrequencies need not be identical between the two types of molecules andcan be varied over time.

In another implementation, shown in example 4, a first type ofion-passing molecule can be activated for a time period followed byactivation of a second type of ion-passing molecule. This can beparticularly useful for allowing each of the ion-passing molecules to beinactive for the time period during which the other ion-passing moleculeis being activated. In certain instances, this can facilitate recoveryand sustained use of the ion-passing molecules. Moreover, the differentcurrent types, densities and ion-passing capabilities can be consideredwhen deciding on the specific stimulation profile to create the desiredresponse.

A specific example of DBS is used for the treatment of Parkinson'sdisease. In this application, DBS is often applied to the globuspallidus interna, or the subthalamic nucleus within a patient's brain.By implanting a biological arrangement that modifies the cells torespond to light, a light flashing light can be used in place ofelectrodes. Thus, the targeted neuron cells and external electricalsignal need not be directly applied to the targeted cells. Moreover,light can often travel from its point of origin farther thanelectricity, thereby increasing the effective area relative to thestimulation source and only those neurons that have been photosensitizedare stimulated.

As with the electrode-based DBS methods, one embodiment of the presentinvention can be implemented using high-frequency DBS to inhibit neurongenerated impulses. While high-frequency DBS has been accomplished atfrequencies around 100 Hz, high-frequency DBS using various embodimentsof the present invention may not necessarily require the same frequency.For instance, it may be possible to reproduce the inhibiting effects ofhigh-frequency DBS at lower frequencies (e.g., 50 Hz) when using lightactivated techniques. For example, activation of the GtR3 pumpintrinsically favors hyperpolarization and resistance to actionpotential generation. Various frequencies can be used depending upon theparticular application (e.g., the targeted portion of the brain and thedesired effect), and the stimulation modality being applied.

Consistent with another example embodiment of the present invention,gene transfer vectors inducing the expression of photosensitivebio-molecules are used to target a specific type of cell. For instance,viral-based proteins (e.g., lentiviruses, adeno-associated viruses orretroviruses) can be created to target specific types of cells, basedupon the proteins that they uniquely express. The targeted cells arethen infected by the viral-based gene-transfer proteins, and begin toproduce a new type of ion channel (for example DChR), thereby becomingphotosensitive. This can be particularly useful for stimulating thetargeted cells without stimulating other cells that are in proximity tothe targeted cells. For example, neurons of disparate length, diameter,chronaxie, other membrane properties, electrical insulation,neurotransmitter output, and overall function, lie in close proximity toone another, and thus, can be inadvertently stimulated when usingelectrodes to provide the stimulation of the neurons. For furtherdetails on the generation of viral vectors and the in vivo modificationand stimulation of neural cells, reference may be made to U.S. patentapplication Ser. No. 11/459,636 filed on Jul. 24, 2006, “An opticalneural interface: in vivo control of rodent motor cortex with integratedfiber optic and optogenetic technology” by Alexander M. Aravanis, et al,Journal Neural Engineering 4 (2007) S143-S156, “Neural substrates ofawakening probed with optogenetic control of hypocretin neurons” byAntoine R. Adamantidis, et al, Nature, (Nov. 15, 2007) Vol. 450:420-424, “Targeting and Readout Strategies for Fast Optical NeuralControl In Vitro and In Vivo” by Viviana Gradinaru, et al, The Journalof Neuroscience, (Dec. 26, 2007) 27(52):14231-14238, “Multimodal fastoptical interrogation of neural circuitry” by Feng Zhang, et al, Nature(Apr. 5, 2007) Vol. 446: 633-639, “Circuit-breakers: opticaltechnologies for probing neural signals and systems” by Feng Zhang, etal, Nature Reviews Neuroscience (August 2007) Vol. 8: 577-581, which areeach fully incorporated herein by reference.

A specific embodiment of the present invention employs an implantablearrangement for in vivo use. A light-emitting diode, laser or similarlight source is included for generating light (as shown, for example,light in FIG. 3). A biological portion that modifies target cells toinclude light responsive molecules which facilitate stimulation of thetarget cells in response to light generated by the light source.

Another embodiment of the present invention employs an arrangement forstimulating target cells using a photosensitive protein that allows thetarget cells to be stimulated in response to light. A biologicaldelivery device, such as those discussed in connection with biologicalportion 204 of FIG. 4, is used for implanting vectors that modify thetarget cells to include the photosensitive protein. An implantationcomponent, such as that discussed in connection with biological portion204, the mesh of FIG. 7 or viral matrix of FIG. 8, is used forimplanting a light generating device near the target cells. A controldevice, such as that discussed in connection control circuit 208, isused for activating the light generating device to generate light to bereceived by the target cells, thereby stimulating the target cells inresponse to the generated light.

Returning now to the figures, FIG. 3 shows a block diagram of a systemfor stimulating target cells, according to an example embodiment of thepresent invention. Block 102 represents a location internal to anorganism (e.g., a mammal), as shown by the in vivo designation. Lightgenerator 104 is an implantable device that generates light in vivo. Thephotosensitive biological portion 106 affects the target cells such thatgenerated light strikes causes stimulation of the target. In oneinstance, the light generator 104 is a small electronic device on theorder of a few millimeters in size. The small size is particularlyuseful for minimizing the intrusiveness of the device and associatedimplantation procedure. In another instance, the light generator 104 mayinclude a fiber optic device that can be used to transmit light from anexternal source to the target cells.

In one embodiment of the present invention, the target cells aremodified to contain light-activated proton pump/channel proteins. Aspecific example of such protein is GtR3, which is a product based uponthe cryptophytes Guillardia theta. Characterization of the actionspectra for GtR3 suggests that the absorption maxima are around 490 nm.Another specific example is DChR from Dunaliella salina. The actionmaxima for DChR are around 500 nm.

These light sensitive proteins can be implanted using a number ofdifferent methods. Example methods include, but are not limited to, theuse of various delivery devices, such as gelatin capsules, liquidinjections and the like. Such methods also include the use ofstereotactic surgery techniques such as frames or computerized surgicalnavigation systems to implant or otherwise access areas of the body. Forfurther details on delivery of such proteins, reference may be made toU.S. patent application Ser. No. 11/459,636 filed on Jul. 24, 2006 andentitled “Light-Activated Cation Channel and Uses Thereof”, which isfully incorporated herein by reference.

FIG. 4 shows a block diagram of an implantable device for stimulatingtarget cells, according to an example embodiment of the presentinvention. The figure includes control circuit 208, light source 206,biological portion 204 and target cells 202. Biological portion 204affects the target cells 202 such that the target cells are stimulatedin response to light

In one embodiment of the present invention, biological portion 204 maybe composed of target cells 202 that have been modified to bephotosensitive. In another embodiment of the present invention,biological portion 204 may contain biological elements such as genetransfer vectors, which cause target cells 202 to become sensitive tolight. An example of this is lentiviruses carrying the gene for DChRexpression. In this manner, the stimulation of target cells 202 can becontrolled by the implantable device. For example, the control circuit208 can be arranged to respond to an external signal by activating, ordeactivating light source 206, or by charging the battery that powerslight source 206. In one instance, the external signal iselectromagnetic radiation that is received by control circuit 208. Forexample, radio frequency (RF) signals can be transmitted by an externalRF transmitter and received by control circuit 208. In another example,a magnetic field can be used to activate and/or power the controlcircuit.

Control circuit 208 can be implemented using varying degrees ofcomplexity. In one instance, the circuit is a simple coil that whenexposed to a magnetic field generates a current. The current is thenused to power light source 206. Such an implementation can beparticularly useful for limiting the size and complexity as well asincreasing the longevity of the device. In another instance, controlcircuit 208 can include an RF antenna. Optionally, a battery or similarpower source, such as a capacitive element, can be used by controlcircuit 208. While charged, the power source allows the circuitry tocontinue to operate without need for concurrent energy delivery fromoutside the body. This can be particularly useful for providing precisecontrol over the light emitted by light source 206 and for increasedintensity of the emitted light. In one embodiment of the presentinvention, light source 206 is implemented using a light-emitting-diode(LED). LEDs have been proven to be useful for low power applications andalso to have a relatively fast response to electrical signals.

In another embodiment of the present invention, biological portion 204includes a gelatin or similar substance that contains gene transfervectors which genetically code the target cells for photosensitivity. Inone instance, the vectors are released once implanted into the body.This can be accomplished, for example, by using a containment materialthat allows the vectors to be released into aqueous solution (e.g.,using dehydrated or water soluble materials such as gelatins). Therelease of the vectors results in the target cells being modified suchthat they are simulated in response to light from light source 206.

In another embodiment of the present invention, the biological portion204 includes a synthetic mesh that contains the photosensitive cells. Inone instance, the cells are neurons that have been modified to bephotosensitive. The synthetic mesh can be constructed so as to allow thedendrites and axons to pass through the mess without allowing the entireneuron (e.g., the cell body) to pass. One example of such a mesh haspores that are on the order of 3-7 microns in diameter and is made frompolyethylene terephthalate. In another example embodiment, thebiological portion 204 includes an injection mechanism as discussed infurther detail herein.

FIG. 5 shows a block diagram of an implantable device, according to anexample embodiment of the present invention. The implantable device ofFIG. 3 is responsive to a field magnetic. More specifically, an inductorconstructed from windings 302 and core 304 generates a current/voltagein response to a magnetic field. The current is passed to controlcircuit 310 through conductive path 306. In response, control circuit310 activates light source 312 using conductive path 308. Light source312 illuminates biological portion 314 in order to stimulate the targetcells. In one instance, biological portion 314 includes a gelatin,synthetic mesh or injection mechanism as discussed in further detailherein.

In one embodiment of the present invention, the control portion can be asimple electrical connection, resistive element, or can be removedcompletely. In such an embodiment, the intensity, duration and frequencyof light generated would be directly controlled by the current generatedfrom a magnetic field. This can be particularly useful for creatinginexpensive, long lasting and small devices. An example of such anembodiment is discussed further in connection with FIG. 6A and FIG. 6B.

In another embodiment of the present invention, the control portion canbe implemented as a more complex circuit. For instance the controlcircuit may include and otherwise implement different rectifiercircuits, batteries, pulse timings, comparator circuits and the like. Ina particular example, the control circuit includes an integrated circuit(IC) produced using CMOS or other processes. Integrated circuittechnology allows for the use of a large number of circuit elements in avery small area, and thus, a relatively complex control circuit can beimplemented for some applications.

In a particular embodiment of the present invention, the inductor (302and 304 of FIG. 5) is a surface mount inductor, such as a 100 uHinductor part number CF1008-103K supplied by Gowanda Electronics Corp.The light generating portion is a blue LED, such as LEDs in 0603 or 0805package sizes. A particular example is a blue surface mount LED havingpart number SML0805, available from LEDtronics, Inc (Torrance, Calif.).Connective paths 306 and 308 can be implemented using various electricalconductors, such as conductive epoxies, tapes, solder or other adhesivematerials. LEDs emitting light in the amber spectrum (as applicable toNpHR channels) and other spectrums are available through commercialsources including this same manufacturer.

FIG. 6A shows a block diagram of an implantable device, according to anexample embodiment of the present invention. FIG. 6A shows an inductorcomprising coils 402 and core 404 connected to LED 408 using conductivepaths shown by 406. FIG. 6B shows a circuit diagram corresponding to theblock diagram of FIG. 6A. Inductor 412 is connected in parallel to LED410. Thus, current and voltage generated by changing a magnetic fieldseen at inductor 412 causes LED 410 to produce light. The frequency andstrength of the changing magnetic field can be varied to produce thedesired amount and periodicity of light from LED 410.

FIG. 7A and FIG. 7B show a diagram of a mesh for containingphotosensitive bio-molecules, according to an example embodiment of thepresent invention. Mesh 502 is constructed having holes 504 of a sizethat allows illumination to pass but is small enough to prevent cells506 to pass. This allows for cells 506 to be implanted while stillreceiving light from a light generator.

In one embodiment of the present invention, the cells 506 are stem cellsthat are modified to be photosensitive. The stem cells are allowed tomature as shown by FIG. 7B. In a particular instance, the stem cellsmature into neurons having a cell body 512, axons/dendrites 508 and 510.The neurons are genetically modified to be photosensitive. Holes 504 areon the order of 3-7 microns in diameter. This size allows some axons anddendrites to pass through holes 504, while preventing the cell body 512to pass.

FIG. 8A and FIG. 8B show a diagram of a viral matrix, according to anexample embodiment of the present invention. The viral matrix includesstructure 602, which contains viral vectors 604. In one instance,structure 602 includes a gel or fluid substance that contains viralvectors 604 until they are implanted in a mammal 606. Once viral vectors604 are released, they infect target cells 608 in the vicinity of theimplanted viral matrix as shown by FIG. 8B. Infected target cell 610becomes photosensitive, and thus, light can be used to control thestimulation of target cell 610.

According to one embodiment of the present invention, structure 602 is agelatin that has been impregnated, or otherwise sealed with viralvectors 604 contained within the gelatin. When structure 602 isimplanted, the gelatin is hydrated and or dissolved, thereby releasingviral vectors 604. Standard commercially available gelatin mix may beused, in addition to compounds such as Matrigel by BD Biosciencesdivision of Becton Dickenson and Company (Franklin Lakes, N.J.)

FIG. 9 shows a circuit diagram of a circuit that produces light inresponse to a magnetic field, according to an example embodiment of thepresent invention. FIG. 9 includes an input circuit 720 and an outputcircuit 730. Inductor 704 generates current in response to magneticfield 702. Due to properties of magnetic fields, the current produced byinductor 704 is an alternating current (AC) signal. Full-wave bridgerectifier 706 rectifies the AC signal and along with an RC circuitgenerates a relatively stable voltage from the AC signal. This generatedvoltage is responsive to magnetic field 702 and output circuit 730 whichgenerates light when the generated voltage is at a sufficient level.More specifically, power from battery 708 is used to drive LED 710 inresponse to magnetic field 702. This is particularly useful forapplications where the magnetic field 702 seen by inductor 704 is lesspowerful (e.g., due to the in vivo location of inductor 704).

FIG. 1 OA shows a circuit diagram of a circuit that produces light inresponse to RF signal 801, according to an example embodiment of thepresent invention. Antenna 802 is used to receive RF transmission 801and convert the signal to electricity. The received transmission isrectified by diode 803 and further filtered by capacitor 805. In a oneinstance, diode 803 can be implemented using a diode having a lowforward bias and fast switching capabilities, such as a Schottky diode.

In a particular embodiment of the present invention, RF transmission 801contains a power component for charging battery 815 and a signalcomponent for controlling LED 825. Capacitor 805 can be selected toseparate these components for use by the circuit. For instance, thepower component may be a relatively low-frequency, large-amplitudesignal, while the signal component is a relatively high-frequency,small-amplitude signal. Capacitor 805 can be selected to filter thepower component of the signal to create a corresponding voltage. Theremaining high-frequency component of the RF transmission is added tothis voltage. The power component of the transmission can then be usedto charge the battery 815, and the signal component of the transmissionis used to enable LED 825. The light generated by LED 825 triggersstimulus of the target cells 827.

FIG. 10B illustrates an alternative embodiment radio-frequency energyaccumulator, which charges a battery, which in turn, powers a digitalpulse generator, which powers a LED. An electromagnetic signal 850 isreceived by loop antenna 852 generating a corresponding electricalsignal. The voltage generated from loop antenna 852 is limited by thereverse bias voltage of the diodes 855 and 856 and stored in capacitor854. In a particular instance these diodes have a low reverse biasvoltage that is relatively precise, such as a Zener diode.Electromagnetic signal 850 is rectified via diode rectifier bridge 858and filtered by voltage regulator 859 to produce a DC voltage. The DCcan be used to charge power source 860.

Battery 860 is coupled to the input of Schmidt trigger 865 throughcapacitor 862. Feedback from the output of the Schmidt trigger isprovided through resistor 864 relative to the charge on capacitor 863.Accordingly, the frequency of the square-wave output of Schmidt trigger865 is determined by the values of the resistor-capacitor networkincluding capacitor 863 and resistor 864. Resistor 864 and capacitor 863may be fixed or variable. The output of Schmidt trigger 865 is fedthrough digital inverter 867 which powers LED 866. Light from LED 866 istransmitted to light-sensitive neurons 868 relative to the frequency ofthe square-wave output of Schmidt trigger 865.

FIG. 10C illustrates block diagram for an electromagnetic field (EMF)energy accumulator and pulsing approach in which the received EMF 897(for example radiofrequency energy) includes not only energy foraccumulation, but also an encoded signal regarding instructions tomicrocontroller 895. In step 885 (Energy plus Parameter Control Signal:Encoding and transmission), a control instruction signal is encoded toride upon the energy component by methods known in the art, for example,by frequency modulation. Energy receiver block 890 uses a portion of theEMF signal to provide power to block 893. Control signal receiver block891 uses a portion of the EMF signal to provide control instructions tomicrocontroller block 895.

The control instruction can be used to transmit information regardingthe various parameters of the generated light, such as frequency,strength, duration, color, and the like. These instructions can bedecoded and processed using a microcontroller or logic circuitry asshown by block 895. Block 895 can generate control signal(s) in responseto the decoded instructions. Accordingly, the frequency (and otherparameters) of the light generated by LED 896 rate need not be fixed forthe given implanted device. Antenna 889 delivers input to the EnergyReceiver 890 (providing power to voltage regulator and battery circuitry893). Concurrently, antenna 889 delivers encoded data to Control SignalReceiver 891, which provides control input to microcontroller 895 thatdrives LED 896. Selected wavelength light 897 is then delivered toelectrically excitable cell 898. The battery in the voltage regulatorand battery circuitry 893 provides power to the microcontroller 895 andthe Control Signal Receiver 891.

The circuit diagrams of FIG. 9 and FIGS. 10A, 10B and 10C are merelyillustrative of a few particular embodiments of the present invention,and various other implementations are envisioned. For example,particular embodiments implement a light source that uses a blue LED;however, other colors and light sources can be implemented dependingupon the particular application. In other particular embodiments thelight source includes more than one color light source. The circuitrycontrols not only when light is provided, but which color, depending onwhether the instructions require inhibition or excitation of the cell.For example, in a specific example the target cell express both aninhibitor (e.g., GtR3) and an exciter (e.g., VChR1), which respond todifferent wavelengths of light. The system can also control the level atwhich the cells are excited or inhibited. This can be done by includingmultiple light responsive proteins in the target cells that excite (orinhibit) and programming the instructions to provide more than one lightwavelength at a single time.

FIG. 11A and FIG. 11B each show a diagram of a fiber-optic device,according to an example embodiment of the present invention. Thefiber-optic device includes a control portion 908, a light generator 906and a fiber optic cable 902.

Fiber optic cable 902 can be positioned near a photosensitive biologicalportion, such as a viral matrix or synthetic mesh as discussed herein.This allows for control portion 908 and light generator 906 to belocated at a distance from the target cells 910 (e.g., at a distancecorresponding to the length of fiber-optic cable 902). This can beparticularly useful for minimizing the size of the portion of theimplanted device that is near the target cells, for example, where thetarget cells are located at or near a sensitive location within thebrain. In some instances, the remote location of portions 908 and 906also facilitates modifications of the device, including, but not limitedto, replacement of various components (e.g., batteries), changes instimulation frequency and length.

Control portion 908 can be configured to respond to an external signal,such as magnetic field or RF signals. Alternatively, control portion 908can be configured to enable light generator 906 according to aprogrammed schedule or a combination of an external signal and aprogrammed response.

FIGS. 12A-12D depicts various stages in the production of aphotosensitive biological portion, according to an example embodiment ofthe present invention. More specifically, FIG. 12A shows moldingstructure 1004 having several molds 1002. Molds 1002 are constructed tovarious sizes depending upon the particular application. In one suchapplication, the molds are a few millimeters or less in diameter.

FIG. 12B shows the molds 1002 from FIG. 12A after applying a layer ofgelatin or similar substance as shown by 1006 and 1008. Moreover, viralvectors (shown by ‘v’) are in the upper two molds. These viruses may besuspended within media 1012, which may be a liquid or gelatinous media.Such liquids include normal saline, HEPES-buffered saline and otherknown viral substances and transfer media. Suitable gelatinous mediaincludes Matrigel (BD Biosciences, San Jose Calif.). These viral vectorsare designed transfer genes for light-sensitization to the membranes oftargeted cells after implantation.

FIG. 12C shows a side view of mold 1006. 1016 represents the moldingstructure that forms the shape of gelatin layer 1014. Gelatin layer 1014traps viral vectors contained within media 1012. A top gelatin layer1010 is applied to fully contain the viral vectors.

FIG. 12D shows the resulting viral vector capsule. The viral vectors1018 are contained within area 1022 by casing 1020. Casing 1020 can bedesigned to dissolve or otherwise allow viral vectors 1018 todisseminate towards the target cells once implanted. In one instance,the capsule is constructed of a water soluble material, for example,gelatin, so that upon implantation the viral vectors are allowed toescape into the body. Water soluble capsule materials are well known inthe pharmaceutical industry.

FIG. 13 shows an implantation device, according to an example embodimentof the present invention. Biological portion 1102 and light generationdevice 1108 are implanted using the implantation device. For example,the shaft of the device 1114 is positioned near the target cells. Next,a user of the device presses on portion 1116 which causes portion 1112to place biological portion 1102 and light generation device 1108 nearthe target cells. The implantation device can then be removed.

FIG. 14A and FIG. 14B show a diagram for another implantation device,according to an example embodiment of the present invention. Implantablelight generating device 1204 is surrounded by, and permeated by fluidchannels 1202. Fluid channels 1202 allow a solution 1210 containingbio-molecular material (e.g., photosensitizing viral vectors) to beinjected immediately proximal to light generating device 1204 and thetarget cells. The fluid channels can be located outside of device 1204and/or within device 1204, as shown by 1212 and 1214 respectively. Inthis manner, the viral vectors can be injected in large quantities orover a period of time. For instance, cells infected by viral vectors canrevert back to their pre-infection state after a period of time. Usingthe device of FIG. 12A, the viral vectors can be periodicallyreintroduced to the target cells. Alternatively, different viral vectorscan be introduced through the fluid channels, allowing for targeting ofdifferent cells at the implantation site. This can be particularlyuseful for staged treatment through stimulation of different types ofcells.

A specific embodiment of the present invention relates to a method forgenetically modifying neurons to express light-sensitive ion channelDChR. In this method, pulses of blue light causes DChR neurons to fireaction potentials corresponding to each pulse. Depolarization andrepolarization occur on a millisecond timescale making this methodconsistent with normal network neurophysiology.

Specific targeted neurons are modified using viral vectors for genetransfer. For further details on the generation of viral vectors,reference can be made to Boyden et al. 2005, Zhang et al. 2006 entitled“Channelrhodopsin-2 and Optical Control of Excitable Cells,” NatureMethods Vol. 3, No. 10, which is fully incorporated herein by reference.This transfection results in the introduction of a gene for a singleprotein, a cell membrane ion channel, known as “DChR”. In nature, DChRresides on the cellular membrane of Dunaliella salina. Upon absorptionof blue/green light (500 nm), this ion channel briefly opens, allowingcation influx. When transfected into a mammalian nerve cell, affectednerves become photosensitive, producing light-triggered actionpotentials.

A neuronal-type specific feature which is also a robust promoter isinserted adjacent to the DChR code within the virus, and the line ispropagated by calcium-phosphate cotransfection of 293FT cells. Thesupernatant is then centrifuged into viral pellets, which are placedwithin phosphate-buffered saline.

In a particular instance, application of a DChR is used for photostimulation. The amino-acid residues comprising DChR channelrhodopsinfrom Dunaliella salina can be used to impart fast photosensitivity uponmammalian nerve cells, by using a viral vector to insert the gene forDChR into targeted nerve cells which may subsequently express this gene.Upon illumination with approximately 500 nm blue/green light, ATRisomerizes and triggers a conformational change to open the channelpore. As DChR is a light-sensitive ion channel, it allows an inwardcurrent to be evoked.

In another instance, application GtR3 (derived from Guillardia theta) isused for photostimulation. This proton pump can be imparted uponmammalian nerve cells by using a viral vector to insert the gene forGtR3 into targeted nerve cells, which may subsequently express thisgene. Upon illumination with approximately 472 nm blue light, activepumping of protons out of the neuronal cytoplasm results inhyperpolarization of the cell.

Since sensitivity to blue light via DChR or GtR3 is induced when a viralvector inserts the respective gene into a previously normal cell, theinsertion may be genetically targeted to the products expressed byspecific cellular subtypes. For example, it might be advantageous tocause only dopaminergic neurons, and not cholinergic neurons to react toblue light.

As discussed above, certain embodiments of the present inventioninvolves the use of an optically responsive ion pump or ion channel thatis expressed in a cell. In a particular instance, the cell is either aneural cell or a stem cell. A specific embodiment involves in vivoanimal cells expressing an ion pump. Certain aspects of the presentinvention are based on the identification and development of a protonpump, derived from Guillardia Theta, for example, for temporally-preciseoptical inhibition of neural activity. The pump allows both knockout ofsingle action potentials within rapid spike trains and sustainedblockade of spiking over many minutes.

According to an example embodiment of the present invention, anoptically responsive ion pump and/or channel is expressed in one or morestem cells, progenitor cells, or progeny of stem or progenitor cells.Optical stimulation is used to activate expressed pumps/channels. Theactivation can be used to control the proton concentration in the cells.It may also be used to control the pH of particular subcellularorganelles. This can be particularly useful for affecting the survival,proliferation, differentiation, de-differentiation, or lack ofdifferentiation in the cells. Thus, optical stimulus is implemented toprovide control over the (maturation) of stem or progenitor cells.

According to other example embodiments of the present invention, methodsfor generating an inhibitory neuron-current flow involve, in a neuron,engineering a protein that responds to light by producing an inhibitorycurrent to dissuade depolarization of the neuron. In one such method,the protein is GtR3 is an exogenous molecule in the neuron, and inanother method the protein is an inhibitory protein that uses anendogenous cofactor.

In another example embodiment, a method for controlling action potentialof a neuron involves the following step: engineering a first lightresponsive protein in the neuron; producing, in response to light, aninhibitory current in the neuron and from the first light responsiveprotein; engineering a second light responsive protein in the neuron;and producing, in response to light, an excitation current in the neuronfrom the second light responsive protein. The combination of lightresponsive proteins may depend on the light sensitivity of the protein,the reaction time of the protein, and defined action of the protein, forexample. Multiple light responsive proteins of the same type (inhibitoryvs. excitatory) may be used in the same cell population to allow for,for example, long term blockage of action potential or single actionpotential inhibition depending on the light wavelength used.

In another method for controlling a voltage level across a cell membraneof a cell, the method comprises: engineering a first light responsiveprotein in the cell; measuring the voltage level across the cellmembrane; and producing, in response to light of a first wavelength andusing the first light responsive protein, a current across the cellmembrane that is responsive to the measured voltage level.

Another aspect of the present invention is directed to a system forcontrolling an action potential of a neuron in vivo. The system includesa delivery device, a light source, and a control device. The deliverydevice introduces a light responsive protein to the neuron, with thelight responsive protein producing an inhibitory current. The lightsource generates light for stimulating the light responsive protein, andthe control device controls the generation of light by the light source.In a particular embodiment the light source is a blue light source andthe light responsive protein is responsive to blue light.

In more detailed embodiments, such a system is further adapted such thatthe delivery device introduces the light responsive protein by one oftransfection, transduction and microinjection, and/or such that thelight source introduces light to the neuron via one of an implantablelight generator and fiber-optics.

Another aspect of the present invention is directed to a method fortreatment of a disorder. The method targets a group of neuronsassociated with the disorder; and in this group, the method includesengineering an inhibitory proteins that use an endogenous cofactor torespond to light by producing an inhibitory current to dissuadedepolarization of the neurons, and exposing the neurons to light,thereby dissuading depolarization of the neurons.

In another aspect of the present invention, GtR3 is optimized formammalian expression. A light responsive protein is isolated fromGuillardia theta. The isolated protein has a C-terminus and anN-terminus. An endoplasmic reticulum (ER) export signal promoter isadded to the C-terminus of the isolated protein, creating an enhancedlight responsive protein. In various embodiments the promoter added tothe isolated GtR3 varies depending on the desired location ofexpression.

FIG. 15 depicts an arrangement with multiple light sources, according toan example embodiment of the present invention. FIG. 15 shows lightsources 1602 and 1604 that illuminate proteins 1610 and 1614. Theproteins 1610 and 1614 are engineered within cell 1612 to controlcurrent across the cell membrane in response to light from light sources1602 and 1604, respectively. In one instance, the first protein 1610functions to dissuade action potential firing, while the second protein1614 functions to encourage action potential firing. Each of proteins1610 and 1614 are responsive to light. In a particular instance, thefirst protein is responsive to light from light source 1602 having awavelength A and the second protein is responsive to light from lightsource 1604 having a wavelength B. Thus, the light sources can be usedto control each protein independently. This can be useful for bothencouraging and dissuading action potentials in the cell. In anotherinstance, having both types of proteins allows for both positive andnegative control of the cell membrane voltage. Thus, the different lightsources and proteins could be used to control the voltage or currentlevel (e.g., clamping) of the cell membrane.

One method of determining responsiveness involves quantifying theresponsiveness in terms of the intensity of light required to produce agiven response. In some instances, the first or second protein can stillbe responsive to the alternate wavelength of light although theresponsiveness of the protein may be less than that of the primarywavelength. Accordingly, a protein of a first type may have someresponsiveness to the wavelength corresponding to the other type ofprotein while still maintaining sufficient independence of operation. Inone such instance, control of the cell can be implemented by shiftingeither the wavelength of light or the intensity of the light. Forinstance, the wavelength can be shifted between A and B to induce acorresponding increase or decrease of the membrane voltage potential.Alternatively, multiple proteins having similar actions, but differentactivation wavelength may be introduced into the same cell. The responsemay vary as the wavelength is shifted, in some places the combination ofthe two responses create a greater response than from an individualprotein.

According to one embodiment of the present invention, pump 1614 canoptionally be implemented for purposes other than dissuading actionpotential firing, such as controlling the voltage level of cell 1612.More specifically, a sensor can be used provide feedback to the lightsource 1602. For instance, this feedback could be a measurement of thevoltage or current across the cell membrane. Thus, the light sourcecould be configured to maintain a constant current or voltage (e.g.,clamp) across the cell. Moreover, the amount of responsiveness can becontrolled by modifying one or more of the intensity and wavelength ofthe light.

FIG. 16 shows a system for controlling electrical properties of one ormore cells in vivo, according to an example embodiment of the presentinvention. Control/Interface unit 1702 enables/disables light source1704 to illuminate target cells 1708. A delivery mechanism, such asfiber optic cable 1706, routes or otherwise directs the light to targetcells 1708. Fiber optic cable 1706 may include a bundle of opticalcables, each capable of carrying and directing light independently.Thus, fiber optic cable 1706 can be configured to deliver light havingone or more wavelengths to multiple locations. Sensor 1710 can beimplemented e.g., as an optical device such as an optical scope or as avoltmeter, to provide feedback to control unit 1702. In a particularinstance, the feedback includes optical imaging of the target cells orof other related cells. In another instance, the feedback could monitorthe voltage response of the target cells, including the amount of actionpotential firing.

FIG. 17 shows a system for controlling electrical properties of one ormore cells in vivo, according to an example embodiment of the presentinvention. Control/Interface unit 1802 enables/disables implantablelight source 1804, which in turn illuminates target cells 1806. Lightsource 1804 is shown with two light source, inhibitory current lightsource 1808 and excitation current light source 1810. Light source 1808produces light at a wavelength and intensity that an inhibitory proteinis responsive to, while light source 1810 produces light at a wavelengthand intensity that an excitation protein is responsive to. One skilledin the art would recognize that various configurations of light source1810 are possible, including a single inhibitory light source or anarray of light sources having one or more wavelengths. Control/Interfaceunit 1802 communicates with light source 1804 through any suitablecommunication mechanisms, such as wired communications or wirelesscommunications using radio-frequency signals, magnetic signals and thelike. As discussed above in connection with FIG. 16, sensor 1812 canoptionally be implemented for providing feedback to control unit 1802.

Certain embodiments of the present invention can be useful in drugscreening. The various light-sensitive proteins, serving to regulatemembrane voltage using ion switches that, when activated (ordeactivated) in response to light, function as channels or pumps and arereferred to hereafter as light-responsive ion switches orlight-activated membrane potential switches (LAMPS).

Consistent with one example embodiment of the present invention, asystem screens for ion-channel and ion-pump affecting compounds. Thesystem introduces one or more drug candidates that could either block orenhance the activity of ion-channels or ion-pumps to cells that weremade optically responsive by the addition of a combination of the abovementioned proteins (ChR2, DChR and NpHR among others), for the purposeof screening the drug candidates. Light triggers optically responsiveion channels in the cells causing a change in the voltage seen acrossthe cell membrane. The voltage change stimulates voltage-gated ionchannels in the cells which will then cause a change in ionconcentrations that can be read as optical outputs. These opticalsignals are detected and used to determine what effect, if any, the drugcandidates have on the voltage-gated ion channels. In a more specificembodiment a protein expressing a proton pump is introduced into thecell.

In one instance, the system allows for different drug candidates to bescreened without necessitating extensive setup between screenings. Forexample, an assay may be performed using optics both to stimulate theoptically responsive cells and to detect the effectiveness of the drug.The use of optics instead of manual contacts, e.g., using a whole-cellpatch clamp, can be particularly useful in increasing the throughput ofthe assay screening process. For instance, the time between screeningscan be reduced by minimizing or eliminating physical manipulationsotherwise necessary to stimulate or detect ion flow in the target cells.The cells can also be prepared prior to the screening process becausethe test equipment need only be optically coupled to the prepared cells.In another instance, throughput may be increased by screening a numberof different drugs simultaneously using, for example, an array of photodetectors and a corresponding array of modified cells exposed todifferent drugs.

A cell line based approach is not limited to a particular ion channel.For example, cell lines can be created for voltage-gated sodium (e.g.,Na_(v)1.1 through Na_(v)1.9), potassium (e.g., K_(v) such as hERG,TASK1, Shaker, or KvLQT1), or chloride conducting channels/pumps (e.g.,members of the CLC family of chloride channels). The methods ofintroducing such genes into the cell line are known in the art and mayinclude, for example liposomal transfection, or viral gene transfer. Forfurther information in this regard, reference may be made to one or moreof the following references:

-   Warren Pear, Transient Transfection Methods for Preparation of    High-Titer Retroviral Supernatants, Supplement 68, Current Protocols    in Molecular Biology, 9.11.1-9.11.18, John Wiley & Sons, Inc.    (1996).-   R. E. Kingston, C. A. Chen, H. Okayama, and J. K. Rose, Transfection    of DNA into Eukarotic Cells. Supplement 63, Current Protocols in    Molecular Biology, 9.1.1-9.1.11, John Wiley & Sons, Inc. (1996).-   R. Mortensen, J. D. Chesnut, J. P. Hoeffler, and R. E. Kingston,    Selection of Transfected Mammalian Cells, Supplement 62, Current    Protocols in Molecular Biology, 9.5.1-09.5.19, John Wiley & Sons,    Inc. (1997).-   H. Potter, Transfection by Electroporation, Supplement 62, Current    Protocols in Molecular Biology, 9.3.1-9.3.6, John Wiley & Sons, Inc.    (1996).-   T. Gulick, Transfection using DEAE-Dextran, Supplement 40, Current    Protocols in Molecular Biology, 9.2.1-9.2.10, John Wile_(y) & Sons,    Inc. (1997).-   R. E. Kingston, C. A. Chen, H. Okayama, Transfection and Expression    of Cloned DNA, Supplement 31, Current Protocols in Immunology (CPI),    10.13.1-10.13.9, John Wiley & Sons, Inc.    Each of the above references is incorporated by reference in its    entirety.

These and other transfer vectors may be generated using various geneticengineering techniques. For instance, the transfer vectors may bederived from a provirus clone of a retrovirus, such as animmunodeficiency virus (e.g., HIV-1 or HIV-2, or SIV). For furtherdetails on the use of 293T cells and transfection thereof, reference canbe made to U.S. Pat. No. 6,790,657 (entitled, Lentivirus Vector System,to Arya), which is fully incorporated herein by reference.

In one embodiment of the invention, optical stimulation of the modifiedcells may be altered to determine specific properties of an introduceddrug candidate. For example, the intensity of the optical stimulus maybe modified to change the corresponding level of depolarization. Thelevel of desired depolarization can be tuned to further characterize theeffectiveness of the drug under test. In another example, the opticalstimulus may include rapid pulsing of the light. By correlating thetemporal relationship between the optical stimulus and the resultantdetected fluorescence, the drug may be further characterized in terms ofa kinetic response. Thus, the drug may be characterized for a variety ofdifferent aspects including, but not limited to, the steady state effecton ion concentrations, a change in the level of depolarization necessaryto trigger the voltage gated ion channels and the effect on repeateddepolarization.

In one embodiment, the system allows for simple calibration of theoptical stimulation and/or detection. The modified cells may beoptically stimulated prior to introduction of the drug candidate. Theion channel responsiveness is detected and recorded. The recorded valuesmay be used as a baseline for comparison to the ion channelresponsiveness of the same modified cells after the introduction of thedrug under test. The recorded values may also be used to modify theoptical stimulus or the sensitivity of the optical detector. Suchmodifications may be applied to an individual test sample or an array oftest samples. For such an array of test samples, each test sample may beindividually calibrated by adjusting the corresponding optical stimulus.Similarly, each corresponding photo detector may be individuallyadjusted.

FIG. 18A shows a basic block diagram of a system for screening forion-channel affecting drugs, according to an embodiment of theinvention. Optical control 1904 communicates with database 1902, opticalsource 1906 and optical detector 1909. Optical source 1906 providesoptical stimulus to test sample 1908. Test sample 1908 includes the drugunder test, cells with optically responsive ion channels, and avoltage/ion indicator. In one instance, the indicator fluoresces inresponse to light from optical source 1906. Optical control 1904 mayalso include a reconfigurable readout, so that as different LAMPS anddifferent LEIAs are used, the same control system can be readily adaptedto each paradigm. Optical detector 1909 produces a signal responsive tosuch florescence, and optical control 1904 receives the produced signal.The optical control 1904 stores data obtained from the signal indatabase 1902. The information stored may include factors such as theintensity, duration and wavelength of the detected light. In aparticular instance, the stored data can be compared against baselinedata, where the baseline data corresponds to data recorded prior to theintroduction of the drug to the test sample 1908. In another instance,optical source 1906 may vary the intensity, duration or other parametersrelated to the control of optical source 1906. These and otherparameters may be stored in database 1902.

It should be apparent that optical source 1906 may be implemented usinga single light source, such as a light-emitting diode (LED), or usingseveral light sources. Similarly, optical detector 1909 may use one ormore detectors and database 1902 may be implemented using any number ofsuitable storage devices.

FIG. 18B shows a system diagram of a large-format, quasi-automatedsystem for drug screening in accordance with a specific embodiment ofthe invention. Control device 1901 (e.g., a computer or control logic)controls various processes, and serves as the central point of systeminput/output functions. The environment may be maintained at anappropriate temperature, humidity, carbon dioxide level and ambientlight level within the walls of the climate control chamber 1905, withthe help of one or more sensors 1914 (e.g., thermostat, carbon dioxidesensor and humidity sensor), carbon dioxide and humidifier apparatus1912, and heater 1910. Multi-well tray 1941 contains test wells 1940 forholding cultured cells, drugs, and other ingredients needed for eachtest. Tray 1941 rests upon X—Y—Z table 1925, the movement of which iscarried out by table actuators 1920, under control of computer 1901.Xenon lamp 1955 emits high-intensity white light 1956, which is passedthrough color filter 1960. In the case that DChR is used for stimulatingthe cells within wells 1940, color filter 1960 is blue, causing bluelight 1961 to exit the filter, and strike dichroic mirror 1970. Bluelight 1961 then passes upward, through microscope objective lensapparatus 1930, and through bottom of transparent tray 1941. In thisfashion, the contents of wells 1940, with their transparent undersides,are illuminated. When a separate wavelength of light is required tostimulate a fluorescent light-emitting indicator of cellular activity, afilter of the appropriate specification may be substituted for theprevious filter 160, causing light of the proper wavelength for thislatter task to be piped toward well 1940. If the cells within well 1940have been light-sensitized, and if the drug being tested in each ofthese wells does not suppress the process, a light-emitting indicator ofcellular activity (LEIA), which has also been added to each well orexpressed by the cells via genetic modification, will emit light inaccordance with the voltage change caused by the effect of the light.This second wavelength of light, which may be much smaller in magnitudethan the stimulation light, is collected by microscope turret 1935, andwill also be passed through dichroic mirror 1975, onto the lens of (CCD)camera 1980.

Dichroic mirror 1970 allows for upward reflection of both the wavelengthrequired to stimulate the optical gating of the membrane (e.g.,blue-green for DChR), and the wavelength required by any LEIA used(e.g., ultraviolet for FURA-2). This dichroic mirror may be arranged toallow passage of the output spectrum of the LEIA (e.g., blue-green forFURA-2) with minimal reflection or absorption.

FIG. 19 is a system diagram of an automated-drug-screening system,according to an example embodiment of the invention. Emitter/detectorunits 2050 make up the emitter/detector array 2051. Emitter/detectorarray 2051 matches the number, size, and layout of the wells on tray2040. Tray holding device 2025 permits tray swapping mechanism 2020 torapidly move a new tray into position once testing of a given tray hasbeen completed. The entire process may be automated, and under thecontrol of device 2001. Device 2001 can be implemented using a computer,control logic, programmable logic arrays, discreet logic and the like.The introduction of the drug candidates under test can also be automatedusing a machine that provides a reservoir for storing the drugs and adispensing nozzle for injecting the drugs into the tray. In a mannersimilar to that shown by FIG. 18, the environment within the walls ofthe climate control chamber 2005 may be maintained at an appropriatetemperature, humidity, carbon dioxide level and ambient light level,with the help of thermostat, carbon dioxide sensor and humidity sensor2014, carbon dioxide and humidifier apparatus 2012, and heater 2010. Theuse of multiple stimulator/detector elements simultaneously and inparallel, can be particularly useful for augmenting the speed of theoverall process. Low cost elements may be used to make multiple paralleldetectors (e.g., the components detailed below in description of FIGS.20A and 20B); the multiple parallel emitter/detector units may also bequite economically feasible.

FIG. 20A depicts the workings of emitter/detector units, such as thoseshown in FIG. 19, according to an example embodiment of the invention.An LED stimulates light-sensitive ion channels of cells located within awell, and a photodiode detects the response of a LEIA. In thisembodiment, device 2101 includes LED 2110, which produces light pulses2111, at the proper wavelength, pulse frequency and intensity, so as tostimulate light-sensitive transgenic cells 2105 in culture within well2106. Due to the presences of an LEIA (e.g., a voltage-sensitive dye ora calcium dye), light 2116 is returned from cells 2105, and is detectedby photodiode 2115. In the case that RH 1691 being used, red light isfluoresced and detected by photodiode 2115. In the absence of cellulardepolarization, no fluorescence is detected by photodiode 2115. Otherlight detecting technologies may also be used instead of a photodiodeincluding phototransistors, and CCD elements.

The combination of photostimulation with optical imaging techniques ofLEIAs may be useful for a number of different reasons. For example,photostimulation may simplify the study of excitable cells by reducingthe need to use mechanical electrodes for stimulation. Severalcommercially available LEIAs are suitable for photogrammetricallyindicating the activation of electrically excitable cells. One such LEIAis calcium dye Fura-2, which may be stimulated with violet/ultravioletlight around 340 nm, and whose fluorescent output is detectable asblue-green light around 535 nm. Another example is voltage sensitive dyeRH 1691, which may be stimulated with green light at about 550 nm, andwhose fluorescent output is detectable as red light at about 70 nm.Another example is voltage sensitive dye di-4-ANEPPS, which isstimulated by blue light at about 560 nm, and whose fluorescent outputis detectable as red light at about 640 nm.

FIG. 20B depicts the workings of another embodiment of theemitter/detector units shown in the FIG. 19, in which multiple effectsare tested within the context of a single well. For example, the cells2155 in the wells 2156 may express both GtR3 and VChR1, and hence besensitive to both the hyperpolarizing effects of blue light, and thedepolarizing effects of amber light. Device 2151 includes LED 2160,which is used for the stimulation of the targeted proton pump (e.g.,GtR3) of light-sensitive transgenic cells 2155. Additional LED 2175 maybe used to stimulate a second targeted ion channel or pump (e.g.,VChR1). Yet another LED 2180 may be used to stimulate a voltagesensitive dye (e.g., RH1691 or calcium dye, such as Fura-2). Each LEDmay be arranged to output specific wavelengths and intensities forstimulus of respective targeted compounds. In one instance, an LED mayaffect more than one target, depending upon the specific sensitivitiesof each compound used. Photodiode 2165 detects the fluorescence of aselected voltage dye, while photodiode 2170 is sensitive to the spectrumfluoresced by a selected calcium dye. The use of multiple LEDs for thesame cell allows for the stimulation of LEIAs at different wavelengths.Multiple LEDs may also be used to detect different light wavelengthsemitted by the LEIA.

FIG. 21A depicts an electronic circuit mechanism for activating the LEDemitters used within the emitter/detector units, according to an exampleembodiment of the invention. Control device 2201 generates a “light onsignal” 2202 to transistor base 2205. This light on signal 2202 willremain on for the duration of a light flash desired, or alternativelymay turn on and off in order to produce rhythmic light flashes at aspecified frequency. Light on signal 2202 permits (conventional) currentto flow from power source 2210, through resister 2211, and throughtransistor collector 2207 and transistor emitter 2212, to ground 2213.Current is also thereby permitted to pass through resistor 2215, andinto LED 2220. LED 2220 emits light 2221, which falls upon well 2225. Ina particular instance, the transistor functions as transconductanceamplifier of signal 2202. In this manner, light of the appropriatewavelength, intensity and frequency is delivered to cells within thewell 2225, so as to cause them to stimulate the particular channel(e.g., DChR) or pump (e.g., GtR3), or other photoactive membranestructure being used to regulate the activity of electrically excitablecells. Various other circuits are also possible. For example, othercircuits can be used in place of circuit 2206 to control LED 2220including, but not limited to, replacing the transistor with anoperational amplifier, a field-effect-transistor, a resistor dividernetwork, transistor-transistor logic, push-pull driver circuits andswitches.

FIG. 21B depicts an example electronic circuit mechanism for lightdetection by the emitter/detector units, according to one embodiment ofthe invention. Control device 2250 may (optionally, depending uponspecific implementation) provide power to photodiode 2255. Photodiode2255 receives fluoresced (emitted) light 2256 from the LEIA on the cellswithin well 2257. The received light results in an output signal. Thisoutput passes through resistor 2260, and is input to Schmitt triggeredhex inverter 2270, which conditions the signal, providing a clean “high”or “low value” to be input to computer 2250.

Operation of the photodetector is shown in photovoltaic mode, but theelement may also be used in the photoconductive mode of operation. Ofcourse, many other light-detection devices and methods may also be used,including phototransistors, photothyristors, and charged-coupled device(CCD) elements, or arrays of elements.

Alternatively, the circuit of FIG. 21B can be used withoutSchmitt-triggered hex inverter 2270, permitting a continuum of signalintensities to be transmitted directly to an analog input to computer2250 or to an analog-to-digital converter. Various other signalconditioning circuits are also possible.

FIG. 22 shows a sequence of steps using the embodiment shown in FIGS.19, 20 and 21, in the context of projected high-throughput process timecourse 2300 and in accordance with one embodiment of the invention. Instep 2305, light of the appropriate wavelength and intensity for thetargeted ion channel is flashed-in this case for approximately threeseconds. Concurrently, a LEIA stimulation flash 2310 may optionally betriggered, depending upon the specific voltage or calcium dye, etc.being used. This LEIA compound may have been previously added to thewell, or may be (artificially) genetically imparted upon the cells suchthat the chemical is produced/expressed by the cells. In step 2315, thelight signal produced by the LEIA is detected by the photodetectorelement (e.g., photodiode). For example, RH1691, fluoresces red light atabout 70 nm.

In step 2320, the signal resulting from the impingement of light ontothe photodetector element is sent back to the computer. This may be abinary (e.g., “high” versus “low” signal intensity), or may be graded toreflect a continuum of activation levels. In the case that multiplephotodetectors are used to determine energies at different wavelengths,the individual readings of these photodetectors may be logged inparallel or in sequence for appropriate interpretation in a later stageof the automated process. In step 2330, the system calls for the nexttray to be placed by the automated system. The next tray is moved intoposition at step 2335 and the process may be repeated until all trays ina batch have been processed.

The amount of time allotted for light delivery may vary, and depends onfactors including the level of light-gated proton or ion channel/pumpexpression, and the density and characteristics of other proton/ionicchannel characteristics of that cell population. The amount of timeallotted for light receipt may vary, and depends upon factors includingthe degree of accuracy required for the screening session. The amount oftime allotted for well-plate (tray) changing may vary, and depends uponfactors including the mechanical speed of the automated apparatus. Iffast neurons are used as the cells being tested, the cellularstimulation and LEIA detection process may be accomplished inmilliseconds.

The process above may be repeated under varying conditions. For example,a given set of cells may be tested with no drug present, andsubsequently with one or more drugs present. The response ofelectrically-excitable cells under those conditions may be therebydocumented, compared and studied. If the invention is implemented withat least one emitter/detector for each well on a tray and at least twoconcurrently operating devices, continuous operation may be maintainedfor extended periods of time.

FIG. 23 illustrates an example of a layout of cell and drug sampleswithin the wells of a well-plate which is suitable for use within anembodiment of the invention. In this figure, well-plate 2401 (alsoreferred to herein as a “tray” contains wells 2405 (examples), which areorganized into columns 2425, labeled with numbers 1-12 and rows 2420,labeled with letters A-H. More specifically, an example column and roware defined by 2410 and 2415 respectively.

As an example of a functional layout of contents introduced into thesewells, rows A-H of a single plate might be used for the testing of twodifferent drugs. To represent a baseline condition, column 1 mightcontain optically gated cells, an endogenous or exogenous LEIA, but nodrug. Columns 2-6 might be used for five different concentrations ofDrug X, one concentration level per column. Likewise, columns 7-11 mightbe use for five different concentrations of Drug Y, one concentrationper column. Column 12, while fully usable, is left unused in thisparticular example.

Variables in the various wells might include the type of cell beingtested, the type of ion channel being tested for, the type of drugplaced in the cell, the concentration of the drug placed in the well,the specific LEIA used, and the optical gating stimulation parameters(e.g., wavelength, intensity, frequency, duration) applied to Inc cellsin that well.

FIG. 24 illustrates the context in which the disclosed invention may beemployed within a larger system which facilitates high-throughput drugscreening. Well-plate 2506 contains wells 2505. These are carriedforward by conveyer 2520, which may be a device such as a conveyor belt,robotic transporter or other delivery mechanism. Pipettes 2510 are heldin array by robotic member 2515, and serve to inject the proper numberof cultured cells and media into wells 2505. Subsequently, well-plate2506 is moved down conveyer 2520, where robotic member 2525, analogousto robotic member 2515 and also containing pipettes, injects the properamount of a LEIA into wells 2505. Conveyer 2520 then brings well-plate2505 into screening chamber 2530. An emitter/detector apparatus, such asthose described in connection with FIG. 17, FIG. 18A, FIG. 18B, FIG.19A, and FIG. 19B, is located within chamber 2530. Additionally,portions of the processes described in FIG. 20A and FIG. 20B may occurwithin this chamber. Subsequently, well-plates 2535 is moved out ofscreening chamber 2530 by conveyor 2540, and discarded at 2545. In analternative embodiment, one or more robotic devices may move pipettes2510, screening chamber 2530, etc. to the locations of well-plate 2506,rather than vice-versa.

Consistent with the above discussion, example screening methods couldinclude the collection of multiple data points without having to switchsamples. Because control over the samples is reversible in the samesample preparation by simply turning the activating light on and offwith fast shutters, the same samples can be reused. Further, a range ofpatterns of stimulation can be provided to the same cell sample so thattesting can be performed for the effect of drugs without concern withregards to differences across different sample preparations. Bymodulating the level of excitation (e.g., by ramping the level from nolight to a high or maximum intensity), the effect of the drug across arange of membrane potentials can be tested. This permits for theidentification of drugs that are efficacious during hyperpolarized,natural, or depolarized membrane potentials.

The cell lines described herein may be a particularly useful fordetailed characterization of drug candidates in a high-throughput mannerOptical control is relatively fast, thereby allowing for the testing thedrug's activity under more physiological forms of activation. Forexample, different frequencies of depolarization and/orhyperpolarization may be used to determine how a drug interacts with thechannel under physiological forms of neural activity. In some instances,the process may be accomplished without the application of expensivechemical dyes to the cell lines.

In conjunction with the various properties discussed herein, the use ofvarious embodiments of the invention may be particularly useful forimproving screening throughput by eliminating the need for cumbersomemechanical manipulation and liquid handling. Various embodiments mayalso be useful for repeatable the screening assay using the samesamples, reducing screening cost by eliminating the need forchemically-based fluorescence reports, producing high temporal precisionand low signal artifact (due to the optical nature of the voltagemanipulation), modulating the level of depolarization by attenuating thelight intensity used for stimulation, and ascertaining the kinetics ofthe drug's modulation on the ion channel through the use of pulsed lightpatterns.

The existence of multiple independently controllable excitation proteinsand inhibition proteins opens the door for a variety of applicationsincluding, but not limited to, applications for treatment of a varietyof disorders and the use of a plurality of light responsive proteinsthat can be selected so as to respond to a plurality of respectiveoptical wavelengths. Although not always expressly stated, inhibitioncan be used in combination with, in addition to, or in place ofexcitation in the applications. The family of single-component proteinshas been shown to respond to multiple wavelengths and intensities oflight. Aspects of the invention allow for further mutations and/orsearches for sequences that allow for additional optical wavelengthsand/or individually controllable protein channels. Variations on theoptical stimulus (e.g., a wavelength, intensity or duration profile) canalso be used. For instance, stimulation profiles may exploit overlaps inthe excitation wavelengths of two different ion channel proteins toallow excitation of both proteins at the same time. In one suchinstance, the proteins may have different levels of responsibility.Thus, in a neural application, one set of ion channels may producespiking at a different success percentage relative to a second set ofion channels. Similarly, the overlaps in inhibition wavelengths of twodifferent ion channels (or pumps) allows for inhibition of both proteinsat the same time. Alternatively, multiple light sources may be usedallowing for stimulations of the light responsive proteins in thecombination desired, while leaving other proteins un-stimulated.

Many human applications of the present invention require governmentalapproval prior to their use. For instance, human use of gene therapy mayrequire such approval. However, similar gene therapies in neurons(non-proliferative cells that are non-susceptible to neoplasms) areproceeding rapidly, with active, FDA-approved clinical trials alreadyunderway involving viral gene delivery to human brains. This is likelyto facilitate the use of various embodiments of the present inventionfor a large variety of applications. The following is a non-exhaustivelist of a few examples of such applications and embodiments.

Addiction is associated with a variety of brain functions, includingreward and expectation. Additionally, the driving cause of addiction mayvary between individuals. According to one embodiment, addiction, forexample nicotine addiction, may be treated with optogeneticstabilization of small areas on the insula. Optionally, functional brainimaging, for example cued-state PET or fMRI, may be used to locate ahyper metabolic focus in order to determine a precise target spot forthe intervention on the insula surface.

Optogenetic excitation of the nucleus accumbens and septum may providereward and pleasure to a patient without need for resorting to use ofsubstances, and hence may hold a key to addiction treatment. Conversely,optogenetic stabilization of the nucleus accumbens and septum may beused to decrease drug craving in the context of addiction. In analternative embodiment, optogenetic stabilization of hyper metabolicactivity observed at the genu of the anterior cingulate (BA32) can beused to decrease drug craving. Optogenetic stabilization of cells withinthe arcuate nucleus of the medial hypothalamus which contain peptideproducts of pro-opiomelanocortin (POMC) andcocaine-and-amphetamine-regulating transcript (CART) can also be used todecrease drug addiction behavior. For further information in thisregard, reference may be made to: Naqvi N H, Rudrauf D, Damasio H,Bechara A. “Damage to the insula disrupts addiction to cigarettesmoking.” Science. 2007 Jan. 26; 315(5811):531-534, which is fullyincorporated herein by reference.

Optogenetic stimulation of neuroendocrine neurons of the hypothalamicperiventricular nucleus that secrete somatostatin can be used to inhibitsecretion of growth hormone from the anterior pituitary, for example inacromegaly. Optogenetic stabilization of neuroendocrine neurons thatsecrete somatostatin or growth hormone can be used to increase growthand physical development. Among the changes that accompany “normal”aging, is a sharp decline in serum growth hormone levels after the4^(th) and 5^(th) decades. Consequently, physical deteriorationassociated with aging may be lessened through optogenetic stabilizationof the periventricular nucleus.

Optogenetic stabilization of the ventromedial nucleus of thehypothalamus, particularly the pro-opiomelanocortin (POMC) andcocaine-and-amphetamine-regulating transcript (CART) of the arcuatenucleus, can be used to increase appetite, and thereby treat anorexianervosa. Alternatively, optogenetic stimulation of the lateral nuclei ofthe hypothalamus can be used to increase appetite and eating behaviors.

Optogenetic excitation in the cholinergic cells of affected areasincluding the temporal lobe, the NBM (Nucleus basalis of Meynert) andthe posterior cingulate gyrus (BA 31) provides stimulation, and henceneurotrophic drive to deteriorating areas. Because the affected areasare widespread within the brain, an analogous treatment with implantedelectrodes may be less feasible than an opto-genetic approach.

Anxiety disorders are typically associated with increased activity inthe left temporal and frontal cortex and amygdala, which trends towardnormal as anxiety resolves. Accordingly, the affected left temporal andfrontal regions and amygdala may be treated with optogeneticstabilization, so as to dampen activity in these regions.

In normal physiology, photosensitive neural cells of the retina, whichdepolarize in response to the light that they receive, create a visualmap of the received light pattern. Optogenetic ion channels can be usedto mimic this process in many parts of the body, and the eyes are noexception. In the case of visual impairment or blindness due to damagedretina, a functionally new retina can be grown, which uses naturalambient light rather than flashing light patterns from an implanteddevice. The artificial retina grown may be placed in the location of theoriginal retina (where it can take advantage of the optic nerve servingas a conduit back to the visual cortex). Alternatively, the artificialretina may be placed in another location, such as the forehead, providedthat a conduit for the depolarization signals are transmitted tocortical tissue capable of deciphering the encoded information from theoptogenetic sensor matrix. Cortical blindness could also be treated bysimulating visual pathways downstream of the visual cortex. Thestimulation would be based on visual data produced up stream of thevisual cortex or by an artificial light sensor.

Treatment of tachycardia may be accomplished with optogeneticstimulation to parasympathetic nervous system fibers including CN X orVagus Nerve. This causes a decrease in the SA node rate, therebydecreasing the heart rate and force of contraction. Similarly,optogenetic stabilization of sympathetic nervous system fibers withinspinal nerves T1 through T4, serves to slow the heart. For the treatmentof pathological bradycardia, optogenetic stabilization of the Vagusnerve, or optogenetic stimulation of sympathetic fibers in T1 through T4will serve to increase heart rate. Cardiac disrhythmias resulting fromaberrant electrical foci that outpace the sinoatrial node may besuppressed by treating the aberrant electrical focus with moderateoptogenetic stabilization. This decreases the intrinsic rate of firingwithin the treated tissue, and permits the sinoatrial node to regain itsrole in pacing the heart's electrical system. In a similar way, any typeof cardiac arrhythmia could be treated. Degeneration of cardiac tissuethat occurs in cardiomyopathy or congestive heart failure could also betreated using this invention; the remaining tissue could be excitedusing various embodiments of the invention.

Optogenetic excitation stimulation of brain regions including thefrontal lobe, parietal lobes and hippocampi, may increase processingspeed, improve memory, and stimulate growth and interconnection ofneurons, including spurring development of neural progenitor cells. Asan example, one such application of the present invention is directed tooptogenetic excitation stimulation of targeted neurons in the thalamusfor the purpose of bringing a patient out of a near-vegetative(barely-conscious) state. Growth of light-gated ion channels or pumps inthe membrane of targeted thalamus neurons is affected. These modifiedneurons are then stimulated (e.g., via optics which may also gain accessby the same passageway) by directing a flash of light thereupon so as tomodulate the function of the targeted neurons and/or surrounding cells.For further information regarding appropriate modulation techniques (viaelectrode-based treatment) or further information regarding theassociated brain regions for such patients, reference may be made to:Schiff N D, Giacino J T, Kalmar K, Victor J D, Baker K, Gerber M, FritzB, Eisenberg B, O'Connor J O, Kobylarz E J, Farris S, Machado A, McCaggC, Plum F, Fins J J, Rezai A R “Behavioral improvements with thalamicstimulation after severe traumatic brain injury,” Nature, Vol. 448, Aug.2, 2007, pp. 600-604.

In an alternative embodiment, optogenetic excitation may be used totreat weakened cardiac muscle in conditions such as congestive heartfailure. Electrical assistance to failing heart muscle of CHF isgenerally not practical, due to the thin-stretched, fragile state of thecardiac wall, and the difficulty in providing an evenly distributedelectrical coupling between an electrodes and muscle. For this reason,preferred methods to date for increasing cardiac contractility haveinvolved either pharmacological methods such as Beta agonists, andmechanical approaches such as ventricular assist devices. In thisembodiment of the present invention, optogenetic excitation is deliveredto weakened heart muscle via light emitting elements on the innersurface of a jacket surround the heart or otherwise against the affectedheart wall. Light may be diffused by means well known in the art, tosmoothly cover large areas of muscle, prompting contraction with eachlight pulse.

Optogenetic stabilization in the subgenual portion of the cingulategyrus (Cg25), yellow light may be applied with an implanted device. Thegoal would be to treat depression by suppressing target activity inmanner analogous to what is taught by Mayberg H S et al., “Deep BrainStimulation for Treatment-Resistant Depression,” Neuron, Vol. 45,651-660, Mar. 3, 2005, pp. 651-660, which is fully incorporated hereinby reference. In an alternative embodiment, an optogenetic excitationstimulation method is to increase activity in that region in a manneranalogous to what is taught by Schlaepfer et al., “Deep Brainstimulation to Reward Circuitry Alleviates Anhedonia in Refractory MajorDepression,” Neuropsychopharmacology 2007, pp. 1-10, which is fullyincorporated herein by reference.

In yet another embodiment, the left dorsolateral prefrontal cortex(LDPFC) is targeted with an optogenetic excitation stimulation method.Pacing the LDLPFC at 5-20 Hz serves to increase the basal metaboliclevel of this structure which, via connecting circuitry, serves todecrease activity in Cg 25, improving depression in the process.Suppression of the right dorsolateral prefrontal cortex (RDLPFC) is alsoan effective depression treatment strategy. This may be accomplished byoptogenetic stabilization on the RDLPFC, or suppression may also beaccomplished by using optogenetic excitation stimulation, and pulsing ata slow rate (e.g., 1 Hz or less) improving depression in the process.Vagus nerve stimulation (VNS) may be improved using an optogeneticapproach. Use of optogenetic excitation may be used in order tostimulate only the vagus afferents to the brain, such as the nodoseganglion and the jugular ganglion. Efferents from the brain would notreceive stimulation by this approach, thus eliminating some of theside-effects of VNS including discomfort in the throat, a cough,difficulty swallowing and a hoarse voice. In an alternative embodiment,the hippocampus may be optogenetically excited, leading to increaseddendritic and axonal sprouting, and overall growth of the hippocampus.Other brain regions implicated in depression that could be treated usingthis invention include the amygdala, accumbens, orbitofrontal andorbitomedial cortex, hippocampus, olfactory cortex, and dopaminergic,serotonergic, and noradrenergic projections. Optogenetic approachescould be used to control spread of activity through structures like thehippocampus to control depressive symptoms.

So long as there are viable alpha and beta cell populations in thepancreatic islets of Langerhans, the islets can be targeted for thetreatment of diabetes. For example, when serum glucose is high (asdetermined manually or by closed loop glucose detection system),optogenetic excitation may be used to cause insulin release from thebeta cells of the islets of Langerhans in the pancreas, whileoptogenetic stabilization is used to prevent glucagon release from thealpha cells of the islets of Langerhans in the pancreas. Conversely,when blood sugars are too low (as determined manually or by closed loopglucose detection system), optogenetic stabilization may be used to stopbeta cell secretion of insulin, and optogenetic excitation may be usedto increase alpha-cell secretion of glucagon.

For treatment of epilepsy, quenching or blocking epileptogenic activityis amenable to optogenetic approaches. Most epilepsy patients have astereotyped pattern of activity spread resulting from an epileptogenicfocus. Optogenetic stabilization could be used to suppress the abnormalactivity before it spreads or truncated it early in its course.Alternatively, activation of excitatory tissue via optogeneticexcitation stimulation could be delivered in a series of deliberatelyasynchronous patterns to disrupt the emerging seizure activity. Anotheralternative involves the activation of optogenetic excitationstimulation in GABAergic neurons to provide a similar result. Thalamicrelays may be targeted with optogenetic stabilization triggered when anabnormal EEG pattern is detected.

Another embodiment involves the treatment of gastrointestinal disorders.The digestive system has its own, semi-autonomous nervous systemcontaining sensory neurons, motor neurons and interneurons. Theseneurons control movement of the GI tract, as well as trigger specificcells in the gut to release acid, digestive enzymes, and hormonesincluding gastrin, cholecystokinin and secretin. Syndromes that includeinadequate secretion of any of these cellular products may be treatedwith optogenetic stimulation of the producing cell types, or neuronsthat prompt their activity. Conversely, optogenetic stabilization may beused to treat syndromes in which excessive endocrine and exocrineproducts are being created. Disorders of lowered intestinal motility,ranging from constipation (particularly in patients with spinal cordinjury) to megacolan may be treated with optogenetic excitation of motorneurons in the intestines. Disorders of intestinal hypermotility,including some forms of irritable bowel syndrome may be treated withoptogenetic stabilization of neurons that control motility. Neurogenicgastric outlet obstructions may be treated with optogeneticstabilization of neurons and musculature in the pyloris. An alternativeapproach to hypomobility syndromes would be to provide optogeneticexcitation to stretch-sensitive neurons in the walls of the gut,increasing the signal that the gut is full and in need of emptying.

In this same paradigm, an approach to hypermobility syndromes of the gutwould be to provide optogenetic stabilization to stretch receptorneurons in the lower GI, thus providing a “false cue” that the gut wasempty, and not in need of emptying. In the case of frank fecalincontinence, gaining improved control of the internal and externalsphincters may be preferred to slowing the motility of the entire tract.During periods of time during which a patient needs to hold feces in,optogenetic excitation of the internal anal sphincter will provide forretention. Providing optogenetic stimulation to the external sphinctermay be used to provide additional continence. When the patient isrequired to defecate, the internal anal sphincter, and then externalanal sphincter should be relaxed, either by pausing the optogeneticstimulation, or by adding optogenetic stabilization.

Conductive hearing loss may be treated by the use of optical cochlearimplants. Once the cochlea has been prepared for optogeneticstimulation, a cochlear implant that flashes light may be used.Sensorineural hearing loss may be treated through optical stimulation ofdownstream targets in the auditory pathway.

Another embodiment of the present invention is directed toward thetreatment of blood pressure disorders, such as hypertension.Baroreceptors and chemoreceptors in regions such as the aorta (aorticbodies and paraaortic bodies) and the carotid arteries (“caroticbodies”) participate in the regulation of blood pressure and respirationby sending afferents via the vagus nerve (CN X), and other pathways tothe medulla and pons, particularly the solitary tract and nucleus.Optogenetic excitation of the carotid bodies, aortic bodies, paraorticbodies, may be used to send a false message of “hypertension” to thesolitary nucleus and tract, causing it to report that blood pressureshould be decreased. Optogenetic excitation or stabilization directly toappropriate parts of the brainstem may also be used to lower bloodpressure. The opposite modality causes the optogenetic approach to serveas a pressor, raising blood pressure. A similar effect may also beachieved via optogenetic excitation of the Vagus nerve, or byoptogenetic stabilization of sympathetic fibers within spinal nervesT1-T4. In an alternative embodiment, hypertension may be treated withoptogenetic stabilization of the heart, resulting in decreased cardiacoutput and lowered blood pressure. According to another embodiment,optogenetic stabilization of aldosterone-producing cells within theadrenal cortex may be used to decrease blood pressure. In yet anotheralternative embodiment, hypertension may be treated by optogeneticstabilization of vascular smooth muscle. Activating light may be passedtranscutaneously to the peripheral vascular bed.

Another example embodiment is directed toward the treatment ofhypothalamic-pituitary-adrenal axis disorders. In the treatment ofhypothyroidism, optogenetic excitation of parvocellular neuroendocrine,neurons in the paraventricular and anterior hypothalamic nuclei can beused to increase secretion of thyrotropin-releasing hormone (TRH). TRH,in turn, stimulates anterior pituitary to secrete TSH. Conversely,hyperthyroidism may be treated with optogenetic stabilization of theprovocellular neuroendocrine neurons. For the treatment of adrenalinsufficiency, or of Addison's disease, optogenetic excitation ofparvocellular neuroendocrine neurons in the supraoptic nucleus andparaventricular nuclei may be used to increase the secretion ofvasopressin, which, with the help of corticotropin-releasing hormone(CRH), stimulate anterior pituitary to secrete ACTH. Cushing syndrome,frequently caused by excessive ACTH secretion, may be treated withoptogenetic stabilization of the parvocellular neuroendocrine neurons ofsupraoptic nucleus via the same physiological chain of effects describedabove. Neuroendocrine neurons of the arcuate nucleus produce dopamine,which inhibits secretion of prolactin from the anterior pituitary.Hyperprolactinemia can therefore be treated via optogenetic excitation,while hypoprolactinemia can be treated with optogenetic stabilization ofthe neuroendocrine cells of the arcuate nucleus.

In the treatment of hyperautonomic states, for example anxietydisorders, optogenetic stabilization of the adrenal medulla may be usedto reduce norepinephrine output. Similarly, optogenetic stimulation ofthe adrenal medulla may be used in persons with need for adrenalinesurges, for example those with severe asthma, or disorders that manifestas chronic sleepiness.

Optogenetic stimulation of the adrenal cortex will cause release ofchemicals including cortisol, testosterone, and aldosterone. Unlike theadrenal medualla, the adrenal cortex receives its instructions fromneuroendocrine hormones secreted from the pituitary and hypothalamus,the lungs, and the kidneys. Regardless, the adrenal cortex is amenableto optogenetic stimulation. Optogenetic stimulation of thecortisol-producing cells of the adrenal cortex may be used to treatAddison's disease. Optogenetic stabilization of cortisol-producing cellsof the adrenal cortex may be used to treat Cushing's disease.Optogenetic stimulation of testosterone-producing cells may be used totreat disorders of sexual interest in women: Optogenetic stabilizationof those same cells may be used to decrease facial hair in women.Optogenetic stabilization of aldosterone-producing cells within theadrenal cortex may be used to decrease blood pressure. Optogeneticexcitation of aldosterone-producing cells within the adrenal cortex maybe used to increase blood pressure.

Optogenetic excitation stimulation of specific affected brain regionsmay be used to increase processing speed, and stimulate growth andinterconnection of neurons, including spurring the maturation of neuralprogenitor cells. Such uses can be particularly useful for treatment ofmental retardation.

According to another embodiment of the present invention, various musclediseases and injuries can be treated. Palsies related to muscle damage,peripheral nerve damage and to dystrophic diseases can be treated withoptogenetic excitation to cause contraction, and optogeneticstabilization to cause relaxation. This latter relaxation viaoptogenetic stabilization approach can also be used to prevent musclewasting, maintain tone, and permit coordinated movement as opposingmuscle groups are contracted. Likewise, frank spasticity can be treatedvia optogenetic stabilization.

In areas as diverse as peripheral nerve truncation, stroke, traumaticbrain injury and spinal cord injury, there is a need to foster thegrowth of new neurons, and assist with their integration into afunctional network with other neurons and with their target tissue.Re-growth of new neuronal tracts may be encouraged via optogeneticexcitation, which serves to signal stem cells to sprout axons anddendrites, and to integrate themselves with the network. Use of anoptogenetic technique (as opposed to electrodes) prevents receipt ofsignals by intact tissue, and serves to ensure that new target tissuegrows by virtue of a communication set up with the developing neurons,and not with an artificial signal like current emanating from anelectrode.

Obesity can be treated with optogenetic excitation to the ventromedialnucleus of the hypothalamus, particularly the pro-opiomelanocortin(POMC) and cocaine-and-amphetamine-regulating transcript (CART) of thearcuate nucleus. In an alternative embodiment, obesity can be treatedvia optogenetic stabilization of the lateral nuclei of the hypothalamus.In another embodiment, optogenetic stimulation to leptin-producing cellsor to cells with leptin receptors within the hypothalamus may be used todecrease appetite and hence treat obesity.

Destructive lesions to the anterior capsule and analogous DBS to thatregion are established means of treating severe, intractableobsessive-compulsive disorder 48 (OCD48). Such approaches may beemulated using optogenetic stabilization to the anterior limb of theinternal capsule, or to regions such as BA32 and Cg24 which showmetabolic decrease as OCD remits.

Chronic pain can be treated using another embodiment of the presentinvention. Electrical stimulation methods include local peripheral nervestimulation, local cranial nerve stimulation and “sub threshold” motorcortex stimulation. Reasonable autogenic approaches include optogeneticstabilization at local painful sites. Attention to promoter selectionwould ensure that other sensory and motor fibers would be unaffected.Selective optogenetic excitation of interneurons at the primary motorcortex also may provide effective pain relief. Also, optogeneticstabilization at the sensory thalamus, (particularly medial thalamicnuclei), periventricular grey matter, and ventral raphe nuclei, may beused to produce pain relief. In an alternative embodiment, optogeneticstabilization of parvalbumin-expressing cells targeting as targetingstrategy, may be used to treat pain by decreasing Substance Pproduction. The release of endogenous opiods may be accomplished byusing optogenetic excitation to increase activity in the nucleusaccumbens. In an alternative embodiment, when POMC neurons of thearcuate nucleus of the medial hypothalamus are optogenetically excited,beta endorphin are increased, providing viable treatment approaches fordepression and for chronic pain.

Certain personality disorders, including the borderline and antisocialtypes, demonstrate focal deficits in brain disorders including“hypofrontality.” Direct or indirect optogenetic excitation of theseregions is anticipated to produce improvement of symptoms. Abnormalbursts of activity in the amygdala are also known to precipitate sudden,unprompted flights into rage: a symptom of borderline personalitydisorder, as well as other conditions, which can benefit fromoptogenetic stabilization of the amygdala. Optogenetic approaches couldimprove communication and synchronization between different parts of thebrain, including amygdala, striatum, and frontal cortex, which couldhelp in reducing impulsiveness and improving insight.

The amygdalocentric model of post-traumatic-stress disorder (PTSD)proposes that it is associated with hyperarousal of the amygdala andinsufficient top-down control by the medial prefrontal cortex and thehippocampus. Accordingly, PTSD may be treated with optogeneticstabilization of the amygdale or hippocampus.

Schizophrenia is characterized by abnormalities including auditoryhallucinations. These might be treated by suppression of the auditorycortex using optogenetic stabilization. Hypofrontality associated withschizophrenia might be treated with optogenetic excitation in theaffected frontal regions. Optogenetic approaches could improvecommunication and synchronization between different parts of the brainwhich could help in reducing misattribution of self-generated stimuli asforeign.

Optogenetic stabilization of cells within the arcuate nucleus of themedial hypothalamus, which contain peptide products ofpro-opiomelanocortin (POMC) and cocaine-and-amphetamine-regulatingtranscript (CART), can be used to reduce compulsive sexual behavior.Optogenetic excitation of cells within the arcuate nucleus of the medialhypothalamus which contain peptide products of pro-opiomelanocortin(POMC) and cocaine-and-amphetamine-regulating transcript (CART) may beused to increase sexual interest in the treatment of cases of disordersof sexual desire. In the treatment of disorders of hypoactive sexualdesire testosterone production by the testes and the adrenal glands canbe increased through optogenetic excitation of the pituitary gland.Optogenetic excitation of the nucleus accumbens can be used for thetreatment of anorgasmia.

The suprachiasmatic nucleus secretes melatonin, which serves to regulatesleep/wake cycles. Optogenetic excitation to the suprachiasmic nucleuscan be used to increase melatonin production, inducing sleep, andthereby treating insomnia. Orexin (hypocretin) neurons strongly excitenumerous brain nuclei in order to promote wakefulness. Optogeneticexcitation of orexin-producing cell populations can be used to treatnarcolepsy, and chronic daytime sleepiness.

Optogenetic stimulation of the supraoptic nucleus may be used to inducesecretion of oxytocin, can be used to promote parturition duringchildbirth, and can be used to treat disorders of social attachment.

Like muscular palsies, the motor functions that have been de-afferentedby a spinal cord injury may be treated with optogenetic excitation tocause contraction, and optogenetic stabilization to cause relaxation.This latter relaxation via optogenetic stabilization approach may alsobe used to prevent muscle wasting, maintain tone, and permit coordinatedmovement as opposing muscle groups are contracted. Likewise, frankspasticity may be treated via optogenetic stabilization. Re-growth ofnew spinal neuronal tracts may be encouraged via optogenetic excitation,which serves to signal stem cells to sprout axons and dendrites, and tointegrate themselves with the network.

Stroke deficits include personality change, motor deficits, sensorydeficits, cognitive loss, and emotional instability. One strategy forthe treatment of stroke deficits is to provide optogenetic stimulationto brain and body structures that have been deafferented from excitatoryconnections. Similarly, optogenetic stabilization capabilities can beimparted on brain and body structures that have been deafferented frominhibitory connections.

Research indicates that the underlying pathobiology in Tourette'ssyndrome is a phasic dysfunction of dopamine transmission in corticaland subcortical regions, the thalamus, basal ganglia and frontal cortex.In order to provide therapy, affected areas are preferably firstidentified using techniques including functional brain imaging andmagnetoencephalography (MEG). Whether specifically identified or not,optogenetic stabilization of candidate tracts may be used to suppressmotor tics. Post-implantation empirical testing of device parametersreveals which sites of optogenetic stabilization, and which areunnecessary to continue.

In order to treat disorders of urinary or fecal incontinence optogeneticstabilization can be used to the sphincters, for example via optogeneticstabilization of the bladder detrussor smooth muscle or innervations ofthat muscle. When micturation is necessary, these optogenetic processesare turned off, or alternatively can be reversed, with optogeneticstabilization to the (external) urinary sphincter, and optogeneticexcitation of the bladder detrussor muscle or its innervations. When abladder has been deafferentated, for example, when the sacral dorsalroots are cut or destroyed by diseases of the dorsal roots such as tabesdorsalis in humans, all reflex contractions of the bladder areabolished, and the bladder becomes distended. Optogenetic excitation ofthe muscle directly can be used to restore tone to the detrussor,prevent kidney damage, and to assist with the micturition process. Asthe bladder becomes “decentralized” and hypersensitive to movement, andhence prone to incontinence, optogenetic stabilization to the bladdermuscle can be used to minimize this reactivity of the organ.

In order to selectively excite/inhibit a given population of neurons,for example those involved in the disease state of an illness, severalstrategies can be used to target the optogenetic proteins/molecules tospecific populations.

For various embodiments of the present invention, genetic targeting maybe used to express various optogenetic proteins or molecules. Suchtargeting involves the targeted expression of the optogeneticproteins/molecules via genetic control elements such as promoters (e.g.,Parvalbumin, Somatostatin, Cholecystokinin, GFAP), enhancers/silencers(e.g., Cytomaglovirus Immediate Early Enhancer), and othertranscriptional or translational regulatory elements (e.g., WoodchuckHepatitis Virus Post-transcriptional Regulatory Element). Permutationsof the promoter+enhancer+regulatory element combination can be used torestrict the expression of optogenetic probes to genetically-definedpopulations.

Various embodiments of the present invention may be implemented usingspatial/anatomical targeting. Such targeting takes advantage of the factthat projection patterns of neurons, virus or other reagents carryinggenetic information (DNA plasmids, fragments, etc), can be focallydelivered to an area where a given population of neurons project to. Thegenetic material will then be transported back to the bodies of theneurons to mediate expression of the optogenetic probes. Alternatively,if it is desired to label cells in a focal region, viruses or geneticmaterial may be focally delivered to the interested region to mediatelocalized expression.

Various gene delivery systems are useful in implementing one or moreembodiments of the present invention. One such delivery system isAdeno-Associated Virus (AAV). AAV can be used to deliver apromoter+optogenetic probe cassette to a specific region of interest.The choice of promoter will drive expression in a specific population ofneurons. For example, using the CaMKIIa promoter will drive excitatoryneuron specific expression of optogenetic probes. AAV will mediatelong-term expression of the optogenetic probe for at least one year ormore. To achieve more specificity, AAV may be pseudotyped with specificserotypes 1, 2, 3, 4, 5, 6, 7, and 8, with each having differenttrophism for different cell types. For instance, serotype 2 and 5 isknown to have good neuron-specific trophism.

Another gene delivery mechanism is the use of a retrovirus. HIV or otherlentivirus-based retroviral vectors may be used to deliver apromoter+optogenetic probe cassette to a specific region of interest.Retroviruses may also be pseudo-typed with the Rabies virus envelopeglycoprotein to achieve retrograde transport for labeling cells based ontheir axonal projection patterns. Retroviruses integrate into the hostcell's genome, therefore are capable of mediating permanent expressionof the optogenetic probes. Non-lentivirus based retroviral vectors canbe used to selectively label dividing cells.

Gutless Adenovirus and Herpes Simplex Virus (HSV) are two DNA-basedviruses that can be used to deliver promoter+optogenetic probe cassetteinto specific regions of the brain as well. HSV and Adenovirus have muchlarger packaging capacities and therefore can accommodate much largerpromoter elements and can also be used to deliver multiple optogeneticprobes or other therapeutic genes along with optogenetic probes.

Focal Electroporation can also be used to transiently transfect neurons.DNA plasmids or fragments can be focally delivered into a specificregion of the brain. By applying mild electrical current, surroundinglocal cells will receive the DNA material and expression of theoptogenetic probes.

In another instance, lipofection can be used by mixing genetic materialwith lipid reagents and then subsequently injected into the brain tomediate transfection of the local cells.

Various embodiments involve the use of various control elements. Inaddition to genetic control elements, other control elements(particularly promoters and enhancers whose activities are sensitive tochemical, magnetic stimulation or infrared radiation) can be used tomediate temporally-controlled expression of the optogenetic probes. Forexample, a promoter whose transcriptional activity is subject toinfrared radiation allows one to use focused radiation to fine tune theexpression of optogenetic probes in a focal region at only the desiredtime.

Parkinson's Disease can be treated by expressing optogeneticstabilization in the glutamatergic neurons in either the subthalamicnucleus (STN) or the globus pallidus interna (GPi) using anexcitatory-specific promoter such as CaMKIIα, and apply optogeneticstabilization. Unlike electrical modulation in which all cell-types areaffected, only glutamatergic STN neurons would be suppressed.

Aspects of the present invention are directed towards testing a model ofa neural circuit or disease. The model can define output response of thecircuit as a function of input signals. The output response can beassessed using a number of different measurable characteristics. Forinstance, characteristics can include an electrical response ofdownstream neurons and/or behavioral response of a patient. To test themodel, optogenetic probes are expressed at an input position for themodel. The optogenetic probes are stimulated and the outputcharacteristics are monitored and compared to an output predicted by themodel.

In certain implementations, the use of optogenetic probes allows forfine tuning of models defined using electrical probes. As electricalprobes provide only limited ability to direct the stimulus and thus arenot well suited for stimulus of certain areas without also directlystimulating nearby areas. Optogenetic probes disclosed herein provide amechanism for more precise selection of the stimulus location. Forinstance, the stimulus from the optogenetic probes can be directed tovery specific types of circuits/cells, such as afferent fibers. Thefollowing description provides an example implementation consistent withsuch an embodiment and is meant to show the feasibility and wide-rangingapplicability for aspects of present invention.

According to one embodiment of the present invention, the invention maybe used in animal models of DBS, for example in Parkinsonian rats, toidentify the target cell types responsible for therapeutic effects (anarea of intense debate and immense clinical importance). This knowledgealone may lead to the development of improved pharmacological andsurgical strategies for treating human disease.

One such application involves long-term potentiation (LTP) and/orlong-term depression (LTD) between two neural groups. By targeting theexpression of VChR1 and ChR2 to different neural populations andstimulating each with a different frequency of light, LTP or LTD can beaccomplished between the two groups. Each group can be individuallycontrolled using the respective wavelength of light. This can beparticularly useful for applications in which the spatial arrangement ofthe two groups presents issues with individual control using the samewavelength of light. Thus, the light delivery device(s) are lesssusceptible to exciting the wrong neural group and can be less reliantupon precise spatial location of the optical stimulus.

The delivery of the proteins to cells in vivo can be accomplished usinga number of different deliver devices, methods and systems. On suchdelivery device is an implantable device that delivers a nucleotidesequence for modifying cells in vivo, such as a viral-vector. Theimplantable device can also include a light delivery mechanism. Thelight delivery can be accomplished using, for example, light-emittingdiodes (LEDs), fiber optics and/or Lasers.

Another embodiment of the present invention involves the use of VChR1 inaffecting stem cell fate including survival/death, differentiation andreplication. The modulation of electrical properties has been shown tocontrol stem cell fate. Various techniques can be used to providestimulus patterns that modify stem cell fate. A specific example isconsistent with the techniques explained in Deisseroth, K. et al.“Excitation-neurogenesis coupling in adult neural stem/progenitorcells,” Neuron 42, pp. 535-552 (2004), which is fully incorporatedherein by reference.

Another embodiment of the present invention is directed to the use ofDChR and/or GtR3 to assess the efficacy of treatments. This can include,but is not limited to, drug screening, treatment regimens or modeling oftreatments/disorders. In a specific embodiment, DChR is used as theprimary optically responsive protein in such assessments. In alternateembodiments, DChR is used with other types of optically responsiveproteins (e.g., VCHR1, GtR3, ChR2 and/or NpHR) that respond to differentwavelengths.

Also provided herein are methods of identifying transsynaptic connectionbetween neuronal cells in an animal or a tissue, comprising: a)administering a first viral vector encoding a Cre recombinase fused to atranscellular tracer protein to neuronal cells in region A of the animalor tissue; b) administering a second viral vector encoding alight-activated protein to neuronal cells in region B of the animal ortissue, wherein the expression of the light-activated protein depends onthe presence of the Cre recombinase; and c) identifying neuronal cellsexpressing the light-activated protein in region B, wherein theexpression of the light-activated protein in the neuronal cellsindicating that these cells are in transsynaptic connection with thecells in region A.

Also provided herein are methods of generating optical control oftargeted neuronal cells in an animal or tissue, comprising: a)administering a first viral vector expressing a Cre recombinase fused toa transcellular tracer protein to region A of the animal or tissue; b)administering a second viral vector encoding a light-activated proteinto region B of the animal or tissue, wherein the expression of thelight-activated protein depends on the presence of the Cre recombinase,and wherein the neuronal cells in region A and in region B are intranssynaptic connected; and c) controlling action potential of aneuronal cell in region B with light that activates the protein.

Also provided herein are methods of controlling action potential of aneuron in an animal, comprising activating a light-activated protein inthe neuron with light to generate action potential change, whereinexpression of the light-activated protein in the neuron is generated bya) administering a first viral vector expressing a Cre recombinase fusedto a transcellular tracer protein to region A of the animal, b)administering a second viral vector encoding a light-activated proteinto region B of the animal which contains the neuron, wherein theexpression of the light-activated protein depends on the presence of theCre recombinase, wherein the neurons in region A and region B are intranssynaptic connection.

In some embodiments, the viral vector is a viral vector selected fromthe group consisting of AAV, HSV, and lentivirus. In some embodiments,the transcellular tracer protein is wheat germ agglutinin (WGA) ortetanus toxin-fragment C (TTC).

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present invention. These and other aspects of theinvention will become apparent to one of skill in the art.

It is understood that aspect and variations of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand variations.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. For example, reference to an “animal cell” is a reference tofrom one to many cells.

An “isolated” polynucleotide is one which has been identified andseparated and/or recovered from a component of its natural environment.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”. The term “about” has its normal meaning of approximately. Insome embodiments, “about” means±10% or ±5%.

TABLE 3 Sequence for GtR3 >GtR3ATGGACTACGGAGGAGCACTGTCTGCTGTGGGCCGTGAATTACTCTTTGTGACCAATCCAGTCGTTGTAAATGGGAGCGTCCTGGTGCCGGAGGATCAATGCTACTGCGCCGGTTGGATTGAAAGCAGAGGCACGAATGGGGCCTCATCCTTCGGCAAGGCCCTACTGGAGTTTGTCTTCATCGTCTTCGCGTGTATCACATTACTGTTGGGAATTAACGCTGCGAAATCAAAGGCTGCATCTAGGGTGCTGTTTCCCGCTACTTTCGTCACTGGAATCGCAAGTATCGCATATTTTTCCATGGCAAGCGGCGGCGGGTGGGTGATTGCCCCTGACTGTCGGCAGCTCTTTGTGGCCCGCTATCTGGACTGGCTCATTACTACACCACTTCTACTCATAGATTTGGGTCTGGTTGCAGGGGTCAGTCGGTGGGATATAATGGCCCTCTGCCTGTCTGATGTCCTGATGATTGCTACGGGTGCTTTCGGGAGCCTGACAGTGGGTAACGTGAAGTGGGTGTGGTGGTTCTTTGGAATGTGTTGGTTTCTTCACATAATCTTCGCGCTTGGGAAAAGTTGGGCAGAAGCAGCCAAGGCCAAGGGCGGCGACTCTGCTTCTGTGTACTCCAAAATCGCCGGCATCACCGTGATTACATGGTTCTGTTATCCCGTGGTATGGGTCTTCGCTGAGGGCTTCGGAAACTTTTCCGTAACCTTCGAAGTTCTCATCTATGGAGTGTTGGATGTTATTTCAAAGGCCGTTTTTGGCCTTATACTGATGTCAGGGGCCGCCACCGGATACGAGTCCATT Translation Map GtR3   1ATGGACTACGGAGGAGCACTGTCTGCTGTGGGCCGTGAATTACTCTTTGTGACCAATCCA   1 M  D  Y  G  G  A  L  S  A  V  G  R  E  L  L  F  V  T  N  P  61GTCGTTGTAAATGGGAGCGTCCTGGTGCCGGAGGATCAATGCTACTGCGCCGGTTGGATT  21 V  V  V  N  G  S  V  L  V  P  E  D  Q  C  Y  C  A  G  W  I 121GAAAGCAGAGGCACGAATGGGGCCTCATCCTTCGGCAAGGCCCTACTGGAGTTTGTCTTC  41 E  S  R  G  T  N  G  A  S  S  F  G  K  A  L  L  E  F  V  F 181ATCGTCTTCGCGTGTATCACATTACTGTTGGGAATTAACGCTGCGAAATCAAAGGCTGCA  61 I  V  F  A  C  I  T  L  L  L  G  I  N  A  A  K  S  K  A  A 241TCTAGGGTGCTGTTTCCCGCTACTTTCGTCACTGGAATCGCAAGTATCGCATATTTTTCC  81 S  R  V  L  F  P  A  T  F  V  T  G  I  A  S  I  A  Y  F  S 301ATGGCAAGCGGCGGCGGGTGGGTGATTGCCCCTGACTGTCGGCAGCTCTTTGTGGCCCGC 101 M  A  S  G  G  G  W  V  I  A  P  D  C  R  Q  L  F  V  A  R 361TATCTGGACTGGCTCATTACTACACCACTTCTACTCATAGATTTGGGTCTGGTTGCAGGG 121 Y  L  D  W  L  I  T  T  P  L  L  L  I  D  L  G  L  V  A  G 421GTCAGTCGGTGGGATATAATGGCCCTCTGCCTGTCTGATGTCCTGATGATTGCTACGGGT 141 V  S  R  W  D  I  M  A  L  C  L  S  D  V  L  M  I  A  T  G 481GCTTTCGGGAGCCTGACAGTGGGTAACGTGAAGTGGGTGTGGTGGTTCTTTGGAATGTGT 161 A  F  G  S  L  T  V  G  N  V  K  W  V  W  W  F  F  G  M  C 541TGGTTTCTTCACATAATCTTCGCGCTTGGGAAAAGTTGGGCAGAAGCAGCCAAGGCCAAG 181 W  F  L  H  I  I  F  A  L  G  K  S  W  A  E  A  A  K  A  K 601GGCGGCGACTCTGCTTCTGTGTACTCCAAAATCGCCGGCATCACCGTGATTACATGGTTC 201 G  G  D  S  A  S  V  Y  S  K  I  A  G  I  T  V  I  T  W  F 661TGTTATCCCGTGGTATGGGTCTTCGCTGAGGGCTTCGGAAACTTTTCCGTAACCTTCGAA 221 C  Y  P  V  V  W  V  F  A  E  G  F  G  N  F  S  V  T  F  E 721GTTCTCATCTATGGAGTGTTGGATGTTATTTCAAAGGCCGTTTTTGGCCTTATACTGATG 241 V  L  I  Y  G  V  L  D  V  I  S  K  A  V  F  G  L  I  L  M 781TCAGGGGCCGCCACCGGATACGAGTCCATT 261  S  G  A  A  T  G  Y  E  S  IRestriction Sites Name Seq. Locations AatI AGGCCT none AccI GTMKAC noneAfIII CTTAAG none AgeI ACCGGT none AlwI GGATC 92 AlwNI CAGNNNCTG noneApaI GGGCCC none ApaLI GTGCAC none AscI GGCGCGCC none AseI ATTAAT noneAvaI CYCGRG none AvaII GGWCC none AvrII CCTAGG none BamHI GGATCC noneBbsI GAAGAC 174(c), 183(c), 678(c) BbvI GCAGC 341, 585, 219(c), 234(c)bclI TGATCA none BglI GCCNNNNNGGC none BglII AGATCT none BlpI GCTNAGCnone BsaI GGTCTC none BsmAI GTCTC none BsmBI CGTCTC none BstEII GGTNACCnone BstXI CCANNNNNNTGG none ClaI ATCGAT none DraIII CACNNNGTG none EagICGGCCG none EarI CTCTTC none EcoRI GAATTC none EcoRV GATATC none FokIGGATG  740, 145(c) FseI GGCCGGCC none HindIII AAGCTT none KasI GGCGCCnone KpnI GGTACC none MluI ACGCGT none NarI GGCGCC none NcoI CCATGG 298NdeI CATATG none NheI GCTAGC none NotI GCGGCCGC none NsiI ATGCAT nonePacI TTAATTAA none PciI ACATGT none PmeI GTTTAAAC none PstI CTGCAG nonePvuI CGATCG none PvuII CAGCTG none SacI GAGCTC none SacII CCGCGG noneSalI GTCGAC none SapI GCTCTTC none SfiI GGCCNNNNNGGCC none SgrAICRCCGGYG none SmaI CCCGGG none SpeI ACTAGT none SphI GCATGC none SspIAATATT none StuI AGGCCT none SwaI ATTTAAAT none TliI CTCGAG none XbaITCTAGA none XhoI CTCGAG none XmaI CCCGGG none XmnI GAANNNNTTC 697 AvaIIIatgcat none AfeI AGCGCT none AvrII CCTAGG none BspEI TCCGGA none BsrGITGTACA none

TABLE 4 Sequence for DChR >DChRATGCGTAGAAGGGAGTCTCAGCTCGCATACCTTTGCCTGTTCGTTTTGATCGCTGGCTGGGCCCCACGTCTGACTGAAAGCGCCCCTGATCTAGCCGAGCGGCGGCCTCCCTCCGAGCGAAACACCCCTTACGCCAATATTAAAAAGGTGCCCAATATAACTGAACCCAACGCCAATGTGCAACTTGATGGGTGGGCTCTGTACCAGGATTTTTACTACCTGGCTGGTTCAGATAAGGAATGGGTCGTTGGCCCTAGCGACCAGTGTTACTGCCGAGCATGGTCTAAATCACACGGCACCGACAGAGAGGGCGAGGCGGCTGTGGTGTGGGCGTACATCGTATTCGCCATTTGTATCGTACAACTGGTTTATTTCATGTTTGCCGCTTGGAAGGCAACGGTCGGATGGGAGGAAGTCTACGTGAACATCATTGAGCTGGTGCACATTGCCCTGGTGATTTGGGTCGAGTTCGATAAACCCGCCATGCTCTACCTTAACGACGGTCAGATGGTTCCATGGTTGCGCTATAGTGCATGGCTCCTTTCCTGCCCAGTCATCCTAATTCACCTGAGCAACTTAACAGGGCTAAAGGGGGACTATAGTAAGAGAACCATGGGGCTTTTGGTCTCTGACATCGGAACCATAGTGTTTGGTACAAGCGCCGCACTCGCTCCGCCAAACCATGTCAAAGTCATCTTATTTACAATTGGGTTGCTGTATGGACTCTTCACTTTTTTCACGGCAGCGAAGGTATATATTGAGGCCTACCACACCGTTCCAAAAGGCCAATGTAGAAACCTCGTGAGGGCTATGGCCTGGACTTATTTCGTAAGTTGGGCGATGTTCCCCATCCTGTTTATCCTGGGAAGAGAGGGTTTTGGCCATATTACATATTTTGGCTCATCCATCGGACACTTCATACTGGAGATATTTTCAAAAAATCTGTGGAGTCTACTGGGCCACGGATTACGGTATCGCATAAGGCAGCATATCATCATTCATGGCAATTTGACAAAGAAGAATAAGATTAATATCGCAGGGGACAACGTCGAAGTGGAAGAGTACGTGGATTCTAACGACAAGGACAGCGACGTT >DChRATGCGTAGAAGGGAGTCTCAGCTCGCATACCTTTGCCTGTTCGTTTTGATCGCTGGCTGGGCCCCACGTCTGACTGAAAGCGCCCCTGATCTAGCCGAGCGGCGGCCTCCCTCCGAGCGAAACACCCCTTACGCCAATATTAAAAAGGTGCCCAATATAACTGAACCCAACGCCAATGTGCAACTTGATGGGTGGGCTCTGTACCAGGATTTTTACTACCTGGCTGGTTCAGATAAGGAATGGGTCGTTGGCCCTAGCGACCAGTGTTACTGCCGAGCATGGTCTAAATCACACGGCACCGACAGAGAGGGCGAGGCGGCTGTGGTGTGGGCGTACATCGTATTCGCCATTTGTATCGTACAACTGGTTTATTTCATGTTTGCCGCTTGGAAGGCAACGGTCGGATGGGAGGAAGTCTACGTGAACATCATTGAGCTGGTGCACATTGCCCTGGTGATTTGGGTCGAGTTCGATAAACCCGCCATGCTCTACCTTAACGACGGTCAGATGGTTCCATGGTTGCGCTATAGTGCATGGCTCCTTTCCTGCCCAGTCATCCTAATTCACCTGAGCAACTTAACAGGGCTAAAGGGGGACTATAGTAAGAGAACCATGGGGCTTTTGGTCTCTGACATCGGAACCATAGTGTTTGGTACAAGCGCCGCACTCGCTCCGCCAAACCATGTCAAAGTCATCTTATTTACAATMGGTTGCTGTATGGACTCTTCACTTTTTTCACGGCAGCGAAGGTATATATTGAGGCCTACCACACCGTTCCAAAAGGCCAATGTAGAAACCTCGTGAGGGCTATGGCCTGGACTTATTTCGTAAGTTGGGCGATGTTCCCCATCCTGTTTATCCTGGGAAGAGAGGGTTTTGGCCATATTACATATTTTGGCTCATCCATCGGACACTTCATACTGGAGATATTTTCAAAAAATCTGTGGAGTCTACTGGGCCACGGATTACGGTATCGCATAAGGCAGCATATCATCATTCATGGCAATTTGACAAAGAAGAATAAGATTAATATCGCAGGGGACAACGTCGAAGTGGAAGAGTACGTGGATTCTAACGACAAGGACAGCGACGTT Translation Map DChR    1ATGCGTAGAAGGGAGTCTCAGCTCGCATACCTTTGCCTGTTCGTTTTGATCGCTGGCTGG    1 M  R  R  R  E  S  Q  L  A  Y  L  C  L  F  V  L  I  A  G  W   61GCCCCACGTCTGACTGAAAGCGCCCCTGATCTAGCCGAGCGGCGGCCTCCCTCCGAGCGA   21 A  P  R  L  T  E  S  A  P  D  L  A  E  R  R  P  P  S  E  R  121AACACCCCTTACGCCAATATTAAAAAGGTGCCCAATATAACTGAACCCAACGCCAATGTG   41 N  T  P  Y  A  N  I  K  K  V  P  N  I  T  E  P  N  A  N  V  181CAACTTGATGGGTGGGCTCTGTACCAGGATTTTTACTACCTGGCTGGTTCAGATAAGGAA   61 Q  L  D  G  W  A  L  Y  Q  D  F  Y  Y  L  A  G  S  D  K  E  241TGGGTCGTTGGCCCTAGCGACCAGTGTTACTGCCGAGCATGGTCTAAATCACACGGCACC   81 W  V  V  G  P  S  D  Q  C  Y  C  R  A  W  S  K  S  H  G  T  301GACAGAGAGGGCGAGGCGGCTGTGGTGTGGGCGTACATCGTATTCGCCATTTGTATCGTA  101 D  R  E  G  E  A  A  V  V  W  A  Y  I  V  F  A  I  C  I  V  361CAACTGGTTTATTTCATGTTTGCCGCTTGGAAGGCAACGGTCGGATGGGAGGAAGTCTAC  121 Q  L  V  Y  F  M  F  A  A  W  K  A  T  V  G  W  E  E  V  Y  421GTGAACATCATTGAGCTGGTGCACATTGCCCTGGTGATTTGGGTCGAGTTCGATAAACCC  141 V  N  I  I  E  L  V  H  I  A  L  V  I  W  V  E  F  D  K  P  481GCCATGCTCTACCTTAACGACGGTCAGATGGTTCCATGGTTGCGCTATAGTGCATGGCTC  161 A  M  L  Y  L  N  D  G  Q  M  V  P  W  L  R  Y  S  A  W  L  541CTTTCCTGCCCAGTCATCCTAATTCACCTGAGCAACTTAACAGGGCTAAAGGGGGACTAT  181 L  S  C  P  V  I  L  I  H  L  S  N  L  T  G  L  K  G  D  Y  601AGTAAGAGAACCATGGGGCTTTTGGTCTCTGACATCGGAACCATAGTGTTTGGTACAAGC  201 S  K  R  T  M  G  L  L  V  S  D  I  G  T  I  V  F  G  T  S  661GCCGCACTCGCTCCGCCAAACCATGTCAAAGTCATCTTATTTACAATTGGGTTGCTGTAT  221 A  A  L  A  P  P  N  H  V  K  V  I  L  F  T  I  G  L  L  Y  721GGACTCTTCACTTTTTTCACGGCAGCGAAGGTATATATTGAGGCCTACCACACCGTTCCA  241 G  L  F  T  F  F  T  A  A  K  V  Y  I  E  A  Y  H  T  V  P  781AAAGGCCAATGTAGAAACCTCGTGAGGGCTATGGCCTGGACTTATTTCGTAAGTTGGGCG  261 K  G  Q  C  R  N  L  V  R  A  M  A  W  T  Y  F  V  S  W  A  841ATGTTCCCCATCCTGTTTATCCTGGGAAGAGAGGGTTTTGGCCATATTACATATTTTGGC  281 M  F  P  I  L  F  I  L  G  R  E  G  F  G  H  I  T  Y  F  G  901TCATCCATCGGACACTTCATACTGGAGATATTTTCAAAAAATCTGTGGAGTCTACTGGGC  301 S  S  I  G  H  F  I  L  E  I  F  S  K  N  L  W  S  L  L  G  961CACGGATTACGGTATCGCATAAGGCAGCATATCATCATTCATGGCAATTTGACAAAGAAG  321 H  G  L  R  Y  R  I  R  Q  H  I  I  I  H  G  N  L  T  K  K 1021AATAAGATTAATATCGCAGGGGACAACGTCGAAGTGGAAGAGTACGTGGATTCTAACGAC  341 N  K  I  N  I  A  G  D  N  V  E  V  E  E  Y  V  D  S  N  D 1081AAGGACAGCGACGTT  361  K  D  S  D  V Restriction Sites Name Seq.Locations AatI AGGCCT 760 AccI GTMKAC 414, 949 AfIII CTTAAG none AgeIACCGGT none AlwI GGATC none AlwNI CAGNNNCTG none ApaI GGGCCC 58 ApaLIGTGCAC 438 AscI GGCGCGCC none AseI ATTAAT 1026 AvaI CYCGRG none AvaIIGGWCC none AvrII CCTAGG none BamHI GGATCC none BbsI GAAGAC none BbvIGCAGC 741, 983 bclI TGATCA none BglI GCCNNNNNGGC none BglII AGATCT noneBlpI GCTNAGC none BsaI GGTCTC 623 BsmAI GTCTC 14, 624 BsmBI CGTCTC noneBstEII GGTNACC none BstXI CCANNNNNNTGG none ClaI ATCGAT none DraIIICACNNNGTG none EagI CGGCCG none EarI CTCTTC 723, 865(c), 1056(c) EcoRIGAATTC none EcoRV GATATC none FokI GGATG 402, 554(c), 848(c), 901(c)FseI GGCCGGCC none HindIII AAGCTT none KasI GGCGCC none KpnI GGTACC noneMluI ACGCGT none NarI GGCGCC none NcoI CCATGG 513, 610 NdeI CATATG none.NheI GCTAGC none NotI GCGGCCGC none NsiI ATGCAT none PacI TTAATTAA nonePciI ACATGT none PmeI GTTTAAAC none PstI CTGCAG none PvuI CGATCG nonePvuII CAGCTG none SacI GAGCTC none SacII CCGCGG none SalI GTCGAC noneSapI GCTCTTC none SfiI GGCCNNNNNGGCC none SgrAI CRCCGGYG none SmaICCCGGG none SpeI ACTAGT none SphI GCATGC none SspI AATATT 135 StuIAGGCCT 760 SwaI ATTTAAAT none TliI CTCGAG none XbaI TCTAGA none XhoICTCGAG none XmaI CCCGGG none XmnI GAANNNNTTC none AvaIII atgcat noneAfeI AGCGCT none AvrII CCTAGG none BspEI TCCGGA none BsrGI TGTACA none

Provided herein is an animal cell including, but not limited to, neuralcells, cell lines and muscle cells, the animal cell comprising: anintegrated exogenous molecule which expresses a proton pump responsiveto blue light, the exogenous molecule derived from Guillardia theta.

Also provided herein is a method comprising: modifying a lightresponsive protein derived from Guillardia theta to add an endoplasmicreticulum (ER) export signal at the C-Terminus of the light responsiveprotein.

Also provided herein is a system for controlling an action potential ofa neuron in vivo, the system comprising: a delivery device thatintroduces a protein to the neuron, the protein being responsive to bluelight, wherein the produces an inhibitory current a blue light sourcethat generates light for stimulation of the blue light responsiveprotein; and a control device that controls the generation of light bythe light source.

Also provided herein are systems, methods, arrangements, or kitsdirected toward: optical stimulation of a cell expressing a GtR3 protonpump.

Also provided herein are systems, methods, arrangements, or kitsdirected toward control of subcellular processes using GtR3 and/or DChR.

Also provided herein are systems, methods, arrangements, or kitsdirected toward the use of GtR3 and DChR in different cell populationsof a common cellular network.

Also provided herein are systems, methods, arrangements, or kitsdirected toward DChR.

Also provided herein is a protein consistent with any of the sequencesdescribed herein (e.g., sequences shown in Table 1).

Also provided herein are systems, methods, arrangements, or kitsdirected toward combinations of DChR or GtR3 with other light-responsiveopsin types, which can be based on reaction profiles thereof.

Also provided herein are systems, methods, arrangements, or kitsdirected toward combinations of three or more light-responsive opsintypes.

Also provided herein are methods for providing tiered-levels of activityusing multiple opsin types in same cell but reactive to different light.

Also provided herein are methods for providing tiered-levels of activityusing multiple opsin types in same cell to provide increase frequency ofchannel function through alternating stimulation of each opsin type.

Also provided herein are systems, methods, arrangements, or kitsdirected toward muscle control.

Also provided herein are systems, methods, arrangements, or kitsdirected toward combination control of several different cell groups,each group having a different combination of opsins so that each groupsbe controlled independently of at least one other group.

Also provided herein is a system comprising a feedback loop for controlof a cell (or cell population) with the cell(s) having multiplelight-responsive proteins types expressed therein.

Also provided herein are therapeutic applications including, but notlimited to, treatment of Parkinson's disease.

Also provided herein are systems, methods, arrangements, or kitsdirected toward implantation in retinal cells/retraining brain torespond to intensity of multiple wavelengths.

Also provided herein are systems, methods, arrangements, or kitsdirected toward drug testing.

Also provided herein are systems, methods, arrangements, or kitsdirected toward the use of GtR3 and/or DChR with transgenic animals.

Also provided herein are systems, methods, arrangements, or kitsdirected toward control of pH levels in cells including, but not limitedto, control over pH levels in an organelle, which can be implemented,without limitation, to encourage or inhibit functions thereof or to killor maim the cell.

EXAMPLES

According to certain embodiments, subcellular and transcellulartrafficking strategies now permit (1) optical regulation at thefar-red/infrared border and extension of optogenetic control across theentire visible spectrum; (2) increased potency of optical inhibitionwithout increased light power requirement (nanoampere-scalechloride-mediated photocurrents that maintain the light sensitivity andreversible, step-like kinetic stability of earlier tools); and (3)generalizable strategies for targeting cells based not only on geneticidentity, but also on morphology and tissue topology, to allow versatiletargeting when promoters are not known or in genetically intractableorganisms. These results illustrate the use of cell-biologicalprinciples to enable expansion of the versatile fast optogenetictechnologies suitable for intact-systems biology and behavior.

Specific aspects of the present disclosure are directed to applicationsof molecular trafficking strategies, to derive a panel of tools thatboth quantitatively and qualitatively enhance the power of optogeneticsand open distinct avenues of investigation. In particular, tools aredeveloped that allow targeting of cells solely by virtue of theirtopological relationships within tissue and that extend the reach ofoptical control to the infrared border, with effector function enhancedbeyond the other known tools and covering the entire visible spectrum.

According to the present disclosure, examination of eNpHR2.0-expressinghippocampal neurons revealed the absence of globular ER accumulationswith persistent intracellular labeling and poor membrane localization,suggesting that additional modifications subsequent to the ER exportstep are important. Examination of primary-sequence differences betweentwo forms of an inward rectifier potassium channel with differentialmembrane localization (Kir2.1 and Kir2.4) revealed differences not onlyin C-terminal ER export motifs but also in N-terminal Golgi exportsignals and in C-terminal trafficking signals (Hofherr et al., 2005).Surprisingly, provision of the Golgi export signal did not significantlyaffect surface expression, but that addition of the trafficking signalfrom Kir2.1 either between eNpHR and the EYFP fusion, or at the Cterminus of the fusion protein, dramatically reduced intracellularlabeling and increased apparent surface membrane expression and alsoimproved labeling of cellular processes. Indeed, high-resolutionconfocal imaging revealed marked localization in processes, withidentifiable labeled membranes spanning intracellular regions apparentlydevoid of the opsin-EYFP fusion protein, in a pattern never previouslyobserved with NpHR or its derivatives.

We examined photocurrents, using whole-cell patch clamp recordings toquantify bona fide functional plasma membrane localization ofhalorhodopsin pump molecules. Photocurrents were indeed profoundlyincreased (to a level ˜20-fold larger than the initially described NpHRcurrents; mean±standard error of the mean [SEM], photocurrent 747.2±93.9pA in lentivirally transduced hippocampal pyramidal neurons under thehuman synapsin I promoter; n=10). FIG. 25D shows representative tracesin the left portion of the figure, and summary plots in the rightportion of the figure. The representative traces and summary plots ofFIG. 25D shows the average photocurrent levels in cells expressingeNPHR3.0 (747.2±93.9 pA), shown in black, and eNpHR2.0, shown in gray,(214.1±24.7 pA; unpaired t test p, 0.0005; n=10). Membrane inputresistance was similar for all neurons patched (eNpHR: 193.1±36.6 Mil;eNpHR3.0: 151.6±28.5 MΩ; unpaired t test p=0.37). At action spectrumpeak described below, nanoampere-scale mean outward currents werereadily observed with 3.5 mW/mm² yellow light, an order of magnitudelower intensity than required by proton pumps to attain this level ofphotocurrent (maintaining low light intensities becomes an importantissue only for in vivo experiments, wherein safe control of significanttissue volumes is paramount) (Aravanis et al., 2007; Adamantidis et al.,2007; Chow et al., 2010). FIG. 25E shows representative voltage traces,in the left portion of the figure, and summary plots, in the rightportion, of eNpHR3.0 (black) and eNpHR2.0 (gray). In virally transducedneurons, light-induced hyperpolarizations by >100 mV were routinelyachievable, at the same modest light power levels, as shown in FIG. 25E(mean hyperpolarization in cells expressing eNpHR3.0: 101.0±24.7 mV,n=10; and eNpHr2.0: 57.2±6.8 mV, unpaired t test p, 0.0005, n=10).Membrane potential changes of this new magnitude represent afunctionally distinct advance in optogenetic inhibition, and weaccordingly designate this third-generation NpHR as eNpHR3.0 (theNatronomonas halorhodopsin was named NpHR in 2005 [Sato et al., 2005],and the first trafficking-enhanced version developed by Gradinaru et al.[2008] is now referred to as eNpHR2.0). As expected from prior work(Zhang et al., 2007a) showing that NpHR photocurrents were step-like andexhibited little inactivation over more than 10 min of continuousillumination (indeed, NpHR was selected for this reason, as described inZhang et al. [2007a]), the eNpHR3.0 photocurrents were also step-like,resistant to inactivation, and highly stable over multiple light pulsesand long (behaviorally relevant) timescales (Zhang et al., 2007a).

FIG. 30A shows stability and recovery for eNpHR3.0 over a short timescale. The representative traces in the left portion of FIG. 30A showphotocurrents in cells expressing eNpHR3.0 when exposed to pairs of 10second long yellow light pulses separated in time by, from the top tobottom of FIG. 30A: 2.5 seconds, 5 seconds, 10 seconds, and 20 seconds.The upper right portion of FIG. 30A shows the summary plot for pulses 20second apart displaying normalized average photocurrent levels in cellsexpressing eNpHR3.0 (P1=first pulse peak, 1.00, S1=first pulse steadystate, 0.74±0.01; P2=second pulse peak, 0.86±0.02; n=11). The lowerright portion of FIG. 30A shows the summary plot for pulses 20 secondsapart displaying approximately 50% peak recovery (P2−S1)/(P1−S1). After20 seconds, the peak recovers to (45.2±6.6)%. FIG. 30B shows thetimecourse of NpHR3.0 normalized photocurrents for long-term continuouslight exposure (n=11; various plotted are mean±SEM). FIG. 30C shows theoutward current of eNpHR3.0 stability over 10 minutes (light deliver of593 nm is indicated by the solid bar; output power density: 2.5 mW/mm²).

To address whether the robust improved expression is preserved in themammalian brain in vivo, we injected lentiviral vectors delivering thenovel opsin gene under control of the CaMKIIα promoter to the CA1 regionof the hippocampal formation in adult mice and examined distribution ofthe expressed EYFP fusion. As in cultured cells, strong expression wasobserved not only in dendrites but also in axons in vivo with botheNpHR3.0 and eNpHR3.1 (a shorter version of eNpHR3.0 with equivalentfunctionality but the N-terminal signal peptide removed). A major invivo opportunity for systems neurobiology is controlling not just aprojection from region A to region B, but a cell type itself that has(among its connections) a projection from A to B. This fundamentallydistinct result requires multiplexing of optical control with othertargeting methods. Such control would be of great value in systemsneurobiology; for example, cortical excitatory pyramidal neurons form agenetically and anatomically defined class of cell, but within thisclass are cells that each project to multiple different areas of thebrain (e.g., thalamus, spinal cord, striatum, and other cortical areas)and therefore have fundamentally distinct roles (Lein et al., 2007;Yoshimura et al., 2005). It is unlikely that genetic tools will advancefar enough to separate all of these different cell classes, pointing tothe need to inhibit or excite cells defined by connection topology (FIG.26B). One way to achieve this goal is to capitalize on transcellulartrafficking: to introduce into the local cell-body location aCre-dependent virus conditionally expressing the microbial opsin gene ofchoice (e.g., Tsai et al., 2009), and rather than additionally employinga Cre-drive mouse line, to instead introduce into a distant targetstructure (chosen to define the cells of interest by anatomicalconnectivity) a virus expressing Cre recombinase fused to atranscellular tracer protein, e.g., wheat germ agglutinin (WGA) (FIG.26B) or tetanus toxin-fragment C (TTC) (Kissa et al., 2002; Maskos etal., 2002; Perreault et al., 2006; Sano et al., 2007; Sugita and Shiba,2005). Cre recombinase in the fusion protein would be transported bypresumed endosomal trafficking mechanisms along with the tracer to thelocal cell-body location if anatomically connected and activate opsinexpression in the subset of local cells defined by this connectivity(FIG. 26B) (Gradinaru et al., 2007, 2009; Petreanu et al., 2007, 2009).Note that this approach does not require any specific promoter fragmentor genetic definition of target cells (a clear advantage for use inless-genetically tractable species such as rats and primates); but, ifneeded, such additional genetic refinements can be readily added (forexample, both the WGA-Cre- and the Cre-dependent opsin could bedelivered under control of cell type-specific promoters whereavailable), creating a versatile means for addressing cells defined atthe intersection of connectivity, location, and genetics.

This concept was first validated in the rat by devising a strategy toselectively introduce eNpHR3.0 into those primary motor cortex (M1)microcircuits that are involved in cortico-cortical connections withprimary sensory cortex (S1) (Colechio and Alloway, 2009). To do this, weinjected the previously described Cre-dependent AAV, now conditionallyexpressing eNpHR3.0 into motor cortex, and injected a novelWGA-Cre-expressing AAV (AAV2-EF1α-mCherry-IRES-WGA-Cre) remotely intoprimary somatosensory cortex. Robust eNpHR3.0-EYFP expression was indeedobserved in a distributed subset of the motor cortex neurons at 5 weeksafter injection, despite the remoteness of the Cre recombinase AAVinjection; in control animals without Cre recombinase, no expression isobserved from these Cre-dependent AAVs (Tsai et al., 2009; Sohal et al.,2009). FIG. 26C shows the construct design for the WGA-Cre and Credependent AAV vectors, wherein the WGA and Cre genes are both optimizedwith mammalian codons. Consistent with the anticipated mode oftrans-synaptic or transcellular transport of Cre, no mCherry-positivecell bodies were observed in motor cortex, and no EYFP-positive cellbodies were observed in S1 sensory cortex. Cre can be trans-synapticallydelivered from transduced cells to activate distant gene expression onlyin synaptically connected neurons that have received the Cre-dependentvirus, and not in others. The expected EYFP-eNpHR3.0 axon terminalsarising from M1 were present in S1. Simultaneous optrodestimulation/recording (Gradinaru et al., 2007) was conducted to validatefunctionality of eNpHR3.0 under the WGA system; indeed, robustinhibition was readily observed in M1, as expected from the intensefluorescence of the XFP-opsin fusion protein. These data indicate thatneurons involved in cortico-cortical connections can indeed be addressedand targeted not simply as a projection, but as a cell type defined byconnectivity.

To independently validate this targeting technology in a distinctcircuit and with a different opsin, we next targeted hippocampalformation dentate gyrus neurons involved in interhemisphericprojections. Within the dentate hilus, the only known monosynapticcontralateral projection arises from the hilar mossy cells, whichterminate on granule cells of the contralateral dentate, in dendrites ofthe molecular layer (Freund and Buzsaki, 1996; Ratzliff et al., 2004).The WGA-Cre AAV was unilaterally injected into one dentate gyrus, whilethe Cre-dependent AAV was injected into the contralateral dentate gyrusof the same animal. Strikingly, opsin expression was observed only inhilar cells of the contralateral side. Indeed, in this case and at thistime point, accumulation of Cre was retrograde and monosynaptic to thecontralateral hilar cells, as no EYFP labeling was observed in thecontralateral granule cell layer; moreover, pointing to lack of directtransduction of axon terminals with this AAV serotype, no mCherry wasobserved in the contralateral dentate. The only EYFP-expressing circuitelements in the ipsilateral dentate, affording precise opportunities foroptical control, were axonal fibers observed to terminate in themolecular layer of the granule cell layer, precisely as expected forfibers arising from the contralateral dentate hilus. Indeed, in vivooptrode recordings confirmed the functionality of WGA/Creactivated ChR2in driving light-triggered spikes both at the opsin-expressing cellbodies and in neurons downstream to the axonal projections ofChR2-expressing cells, in the contralateral hemisphere; in line withprevious optogenetic studies (Zhang et al., 2007a, 2007b), 470 nm lightpulses at 30 Hz (5 ms pulse width) delivered through the optical fiberreliably drove neuronal firing in vivo.

The utility of these targeting strategies for engineered opsins withinintact tissue raised the question of whether additional advantages mightaccrue with regard to volume of tissue modulatable in vivo. Whilemembrane trafficking modifications alone will not shift the actionspectrum, the capability to control neurons in the far red is along-sought goal of optogenetics, as this will allow use of light thatpenetrates much more deeply into scattering biological tissues (as withthe far-red utility recently demonstrated for fluorescent proteins) (Shuet al., 2009), and therefore will allow recruitment of larger volumes(Aravanis et al., 2007; Adamantidis et al., 2007; Gradinaru et al.,2009). The massive photocurrents observed for eNpHR3.0 (˜20× thoseinitially reported for NpHR, which itself is capable of blocking spikingin response to 589 nm amber light), suggested optogenetic control withfar-red light might be achieved. We therefore explored optical controlin the far red with the trafficking-enhanced eNpHR3.0.

Even in response to true-red (630 nm) light of only 3.5 mW/mm², weobserved potent ˜250 pA outward photocurrents in virally transducedcells-still more than 6-fold larger than the first observed NpHRcurrents with yellow light (FIG. 27A), and maintaining thecharacteristic step-like, stable kinetics typical of NpHR(eNpHR3.0-expressing and eNpHR2.0 expressing neurons: eNpHR3.0:239.4±28.7 pA; eNpHR2.0: 42.7±4.5 pA; unpaired t test p=0.00004; n=10)(Zhang et al., 2007a). Moreover, we found that these photocurrentsevoked by red light could be used to trigger large (>40 mV)hyperpolarizations in hippocampal pyramidal neurons (FIG. 27B)(eNpHR3.0-expressing and eNpHR2.0 expressing neurons: eNpHR3.0: 43.3±6.1mV; eNpHR2.0: 15.6±3.2 mV; unpaired t test p=0.00116; n=10). Wetherefore explored even further red-shifted light. We continued toobserve robust photocurrents in the deep red with 660 nm light and atthe red/infrared border with 680 nm light (FIG. 27C). At 680 nm, thephotocurrents (˜75 pA) were still larger than peak (yellow light)eNpHR2.0 currents previously reported at 7 mW/mm². Importantly, at allof the red and far-red wavelengths tested, eNpHR3.0 photocurrentsreadily blocked action potentials induced by current injection (FIG.27D) with 7 mW/mm² or less, validating the extension of optogeneticcontrol channels to far-red light. The outward photocurrents evoked bydifferent wavelengths of red and far-red/infrared border illuminationare: 239.4±28.7 pA (n=10) at 630 nm; 120.5±16.7 pA (n=4) at 660 nm; and76.3±8.1 pA (n=4) at 680 nm.

One important feature of NpHR is its spectral compatibility with ChR2:the two opsins have largely separable action spectra and operate withsimilar light power density requirements, allowing bidirectional controlof optical activity (Zhang et al., 2007a) in vitro or in vivo despite asmall region of spectral overlap. To test whether eNpHR3.0 had becometoo potent, given the spectral overlap, to use in combination with ChR2in the same cell, we created a bicistronic vector containing eNpHR3.0;analogous 2A-based combination vectors (Ryan and Drew, 1994) have beenemployed with earlier tools, and channelrhodopsin currents with thismethod have ranged from 150 to 240 pA and halorhodopsin currents from 11to 40 pA (Tang et al., 2009, Han et al., 2009b). We transfected theeNpHR3.0-2A-ChR2 construct (abbreviated eNPAC) into hippocampalpyramidal neurons. With these experiments, trafficking of both opsingene products to cellular processes was observed. To verify thatindependent excitation and inhibition was still possible despite theincreased currents from eNpHR3.0, we mapped out the steady-statephotocurrent action spectrum in detail for eNPAC and for ChR2(H134R)(Gradinaru et al., 2007) and eNpHR3.0 alone. FIG. 27F, shows theactivation spectrums for eNPAC, the left portion of FIG. 27F, and forChR2 (H124R) and eNpHR3.0, on the right portion of FIG. 27F. In theright portion of FIG. 27F, the activation spectrum of ChR2 is shown indark gray, and the activation spectrum of eNpHR3.0 is shown in lightgray. Maximal eNPAC steady-state excitatory and inhibitory currents wereboth approximately 60% of that observed when each opsin was expressedindividually, yielding maximal photocurrents of >550 pA in eachdirection (FIGS. 27F-G); the modestly overlapping action spectra mayprovide a feature, in that potent shunting inhibition combined withhyperpolarizing inhibition is likely possible with this combinationapproach. More specifically, maximum eNPAC steady-state excitation was567±49 pA at 427 nm (n=9), 62% of the value for ChR2(H134R) alone(916±185 pA, n=5). Similarly, maximum eNPAC inhibition was 679±109 pA at590 nm (n=9), 61% of the value for eNpHR3.0 alone (1110±333 pA; n=4).Output power density for peak current values was 3.5-5 mW/mm² (3.5mW/mm² at 590 nm). Validation in vivo will require demonstration thatthe specific P2A method (or other linker approach) is functional in aparticular circuit or cell type (yet to be determined); however, thesedata in cultured hippocampal neurons demonstrate that potentbidirectional photocurrents of >500 pA each can be achieved within asingle cell, without incapacitating interference of thetrafficking-enhanced opsin.

The known wide action spectrum of the microbial opsins poses challengeswith regard to achieving multiple independent channels of control;interestingly, eNpHR3.0 becomes not only a potent far-red opticalcontrol tool, but also the most potent known blue light-drivenopsin-based inhibitor (>400 pA at 472 nm). Indeed, the membranetrafficking strategies delineated here may form a generalizable strategyfor adapting diverse microbial opsins with unique properties foroptogenetic control purposes. In a final series of experiments, weexplored whether these and other enhanced membrane traffickingprinciples could enable the addition of genetically and functionallydistinct components to the optogenetic toolbox.

While a very large number of microbial opsins genes exist in nature, weand others have thus far found none that outperform eNpHR3.0 (asdescribed herein) with regard to photocurrent size, light requirements,or kinetics (Zhang et al., 2007a; Han and Boyden, 2007; Chow et al.,2010). It is important to continue to expand the optogenetic toolbox,but we have found that most microbial opsins traffic poorly in mammaliancells. However, application of the trafficking principles for microbialopsin engineering outlined here may enable optogenetics to continue thegenomics progress over the past few years (Zhang et al., 2008),capitalizing on the immense natural diversity of microbial opsins (Zhanget al., 2008; Chow et al., 2010). We sought to test the adaptability ofthe membrane trafficking principle with the best-characterized microbialopsin, bacteriorhodopsin (BR) (Stoeckenius and Bogomolni, 1982), from H.salinarum, a green light-activated regulator of transmembrane ionconductance (Marti et al., 1991).

We found that expressed in unmodified form, prominent intracellularaccumulations were observed, similar to those seen when the Natronomonashalorhodopsin is expressed at high levels, and no photocurrents wereobserved. However, addition of the trafficking signal (TS, as employedfor eNpHR3.0) between BR and EYFP substantially improved membrane andprocess localization, with smaller persistent ER-like accumulations thatwere eliminated with further C-terminal addition of the ER export signalFCYENEV. The resulting construct (eBR, doubly engineered for optimalmembrane trafficking) was well tolerated in cultured neurons, withmarked membrane localization and process targeting. Validation offunctional plasma membrane targeting revealed that eBR could typicallydeliver ˜50 pA of outward photocurrent and ˜10 mV hyperpolarizationsthat sufficed to block spiking in hippocampal pyramidal neurons whenexposed to the optimal wavelength light of 560 nm, thereby providinganother channel for optogenetic control and illustrating the potentialgeneralizeability of the microbial opsin membrane trafficking approach.More specifically, as seen in FIG. 28B, five hundred sixty nanometerlight induced outward photocurrents of 46.4±7.2 pA in eBR cells(mean±SEM is plotted, n=12). The membrane input resistance was similarfor all neurons patched (131.6±19.5 me). The light power density atsample was 7 mW/mm² As seen in FIG. 28C, the light inducedhyperpolarizations were 10.8±1.0 mV (mean±SEM plotted, n=12).

We also continued genomic strategies similar to those that allowed ouridentification of the red-shifted excitatory opsin VChR1 from Volvoxcarteri (Zhang et al., 2008); indeed, a number of microbes have beenreported to display light sensitivities from violet to near infrared. Weaccordingly continued our broad genomic mining approach in environmentalsequencing databases, plant/microbial expressed sequence tag (EST)libraries, and whole-genome shotgun (WGS) sequencing repositories tosearch for new rhodopsins with channel or pump properties and novellight sensitivities (Zhang et al., 2008). Using the primary amino acidsequences for ChRs, HRs, and BRs as the template sequence, we continuedthe search among evolutionarily distant species (Zhang et al., 2008;Chow et al., 2010). Among other candidate sequences from diverse hosts(Cryptomonas, Guillardia, Mesostigma, Dunaliella, Gloeobacter, etc.),one of these from Guillardia theta was different from the previouslyreported GtR1 and GtR2 (Sineshchekov et al., 2005) and showed high aminoacid homology to ChR2. We designated this new protein as G. thetarhodopsin-3 (GtR3), optimized the codon bias of GtR3 for mammalianexpression, and delivered the GtR3-EYFP fusion gene to hippocampalpyramidal neurons. In an emerging theme, GtR3 showed intracellularaccumulations and no photocurrents. Provision of the TS signal betweenGtR3 and EYFP only mildly reduced accumulations, but, together withaddition of the ER export signal FCYENEV to the C terminus,accumulations were abolished and increased surface and processlocalization observed.

The resulting modified GtR3 hyperpolarizes hippocampal neurons inresponse to 472 nm blue light, albeit with smaller currents than eBR,and could inhibit spiking as well. We also achieved blue inhibition ofspiking with an opsin (AR) from Acetabularia acetabulum (Tsunoda et al.,2006; Chow et al., 2010) engineered for improved trafficking; ARgenerates little current alone but was initially aggregate free andrequired only addition of the TS signal between AR and EYFP forfunctional membrane localization and spike inhibition. Sample currentclamp and voltage clamp traces and summary data show GtR3 function under472 nm light (18.5 mW/mm²) are shown in the left and right portions,respectively, of FIG. 31C. A light induced outward photocurrent summaryis shown in the left bar graph of FIG. 31C, and the correspondinghyperpolarization summary for a blue light peak is shown in the rightbar graph. Corresponding photocurrents and hyperpolarization were:0.5±0.4 pA and 0.12±0.09 mV for yellow light (589 nm; 7.5 mW/mm²);20.0±6.7 pA and 5.6±1.2 mV for blue light (472 nm; 18.5 mW/mm²); 1.7±0.9pA and 0.6±0.3 mV for purple light (406 nm; 3 mW/mm²) (mean±SEM plotted;n=10; input resistance was similar for all neurons: 113.5±24.2 mΩ).

While these and other published microbial opsin-derived inhibitors arenot yet as potent as eNpHR3.0 (and for this reason we continue witheNpHR3.0 for optogenetic applications), the improved functionalityachieved here by membrane trafficking modifications point to thepotential versatility of this approach in unlocking the full potentialof ecological diversity, and to the individualized strategies that willbe indicated for different microbial opsin genes. FIG. 29A shows generalsubcellular targeting strategies for adapting microbial opsin genes tometazoan intact-systems biology. FIG. 29B shows refinement of targetingat the tissue and subcellular levels (subcellular opsin targetingmethods have been previously described, see Gradinaru et al. [2007] andLewis et al., [2009]; tissue/transcellular opsin targeting methods aredescribed herein).

Optogenetic approaches previously have found substantial utility in thecontrol of biological processes and behavior in freely moving mammals(Adamantidis et al., 2007; Airan et al., 2009; Gradinaru et al., 2009;Petreanu et al., 2007, 2009; Sohal et al., 2009; Tsai et al., 2009) andother animals (Douglass et al., 2008; Hwang et al., 2007; Lerchner etal., 2007; Zhang et al., 2007a), with the high temporal precision thatis important for intact-systems biology. We have found that engineeringspecific membrane-trafficking capabilities for microbial proteins is animportant step in generating diverse optogenetic technologies forintact-systems biology. Not all trafficking strategies will be suitablefor all microbial opsins, with different motifs required for opsins thatencounter trafficking difficulty at different stages; therefore, carefulsubcellular analysis with rational selection of proper modificationstogether constitute a directed and principled strategy that may beapplicable to all opsins of nonmammalian origin, thereby enablingsystematic generation of novel optogenetic tools from genomic resources.

We have previously observed that inhibition with NpHR and NpHR2.0, whileuseful for many applications (Gradinaru et al., 2009; Sohal et al.,2009; Tønnesen et al., 2009; Arrenberg et al., 2009), can in some casesbe overcome by very strong excitation (Sohal et al., 2009).Hyperpolarizations by greater than 100 mV with eNpHR3.0 provide asubstantial step forward in the potency of optical inhibition. Theinhibition now provided with eNpHR3.0, more than 20-fold stronger thanthe initial NpHR, remains tunable with light intensity or duty-cycleadjustments, as with any of the optogenetic tools. At the actionspectrum peak, nanoampere-scale mean outward currents readily resultedwith only 3.5 mW/mm² yellow light (10-fold less light power thanrequired for approaching similar currents with previously describedproton pumps). At the same time, eNpHR3.0 preserved the step-likekinetics, fast recovery, and resistance to inactivation over longtimescales of NpHR (Zhang et al., 2007a).

eNpHR3.0 is particularly well suited for in vivo applications, as themost red-shifted and potent optogenetic inhibitor to date, but furtherstrategies to enhance potency of this and other tools will no doubtemerge, and membrane trafficking work as described here may enable evenmore potent inhibitors in the future. When employing eNpHR3.0,inhibition can be readily dialed down if needed by using weakerpromoters, shorter expression times, or reduced light levels, whilemaintaining access to new wavelengths ˜100 nm red-shifted from previousreports (680 nm versus 589 nm) to enable operating at the infraredborder with deeper-penetrating and safer photons. Of course, not onlythe opsin gene and light source selected, but also the strategy forcircuit element targeting, may determine effectiveness; for example,optogenetic work on Parkinsonian models (Gradinaru et al., 2009) showedthat therapeutically effective deep brain stimulation (DBS) in thesubthalamic nucleus (STN) is likely initiated by action on afferentaxons (which in turn then modulate both downstream and upstreamnetworks). While direct fiber-optic-based inhibition of local cellbodies in the STN did not show behavioral effects comparable to thoseobserved with direct modulation of afferent axons, these results do notmean that inhibition of the STN is not important (indeed, optogeneticaxonal modulation in the STN results in inhibition of STN spiking, asnoted by Gradinaru et al. [2009]). Rather, these results informed thelong-standing clinical significant questions surrounding the mechanismand target of DBS by showing that axonal modulation constitutes thelikely therapeutic mechanism and a highly efficient means for a pointsource such as a DBS electrode (or optical fiber) to control a structureor network in diseased neural circuitry (Gradinaru et al., 2009). Inaddition to these circuit element targeting considerations, the lightintensity and wavelength, promoter choice, virus titer, virus tropism,time of opsin expression, target cell biophysics, and local patterns ofendogenous activity and modulation will all affect optogeneticinhibition efficacy and should be carefully considered for eachexperimental system (Cardin et al., 2010).

To enable control of cell types on the basis of connectivity properties,we leveraged transcellular delivery of a Cre recombinase. First, in rat,we selectively targeted those M1 neurons that are involved incortico-cortical connections with S1, and second, we targetedhippocampal formation dentate gyrus neurons involved in interhemisphericprojections; in both cases, cells were targeted defined only byconnectivity, without use of cell type-specific promoter fragments ortransgenic animals. In each system, this approach must be validated fordirectionality (antero- or retrograde) and extent of Cre transport,which may depend on cell-specific endosomal dynamics and experimentaltimepoint; this strategy may also work only with viruses rather thanmouse transgenesis, allowing Cre access to episomal DNA. This approachis best served by vectors that do not directly transduce axon terminals,which may not be the case for all AAV serotypes (as we and others havein some circuits observed [Paterna et al., 2004]). Any directtransduction of axon terminals may be detected or ruled out withappropriate XFP markers, and direct transduction of axon terminals insome cases may be desirable and can be achieved with HSVs, some AAVserotypes, and pseudotyped lentiviruses; however, such an approach(unlike the present strategy) does not allow selection of the cell typepostsynaptic to the transduced terminals and is not as efficient withcertain AAVs that are the vector of choice for many applications due tohigh titer, tissue penetration, safety, and tolerability.

The cell-process targeting enabled by membrane trafficking modificationallows for control of cells that are defined topologically—that is, bythe essential character of their connections within tissue. At present,it is not guaranteed that transport is synaptic or monosynaptic (as inthe hippocampal circuit experiments, such properties will need to bevalidated in each system); therefore, the term “topological targeting”rather than synaptic targeting is here used to underscore thedeformation independence of the fundamental character of theconnection—an axonal connection can take any path from A to B, and aslong as the connection is present, the topological targeting strategyremains valid. This property is important in genetically less-tractableorganisms, but also of substantial value even in animals such as mice,where genetic targeting tools are in many cases inadequate. Of course,genetic targeting strategies may be multiplexed with topologicaltargeting; for example, expression from the Cre-dependent vector and theCre-fusion vector may each be governed by specific genetic targetingsequences if available. Moreover, the availability of multiple channelsof optical control opens the door to combinatorial topological targetingstrategies.

Like ChR2, NpHR, and VChR1, we note that most microbial opsins canbenefit from substantial protein engineering to achieve new kinds offunctionality. Indeed, we and others have previously demonstratedmolecular strategies for eliciting from microbial opsins increased lightsensitivity (Berndt et al., 2009), increased photocurrent magnitude(Gradinaru et al., 2007, 2008; Zhao et al., 2008), faster kinetics(Gunaydin et al., 2010; Lin et al., 2009), and bistable switchingbehavior (Berndt et al., 2009). Other possibilities such as shiftedaction spectrum (Zhang et al., 2008; Gunaydin et al., 2010), increasedtwo-photon responsivity, and altered ion permeability (e.g., for Ca2+)may also be achieved in the future.

While ion conductance-regulating opsins have been the most versatile forready translation (employing a common electrical language), biochemicalcontrol with light in defined cell types is also possible (but with adifferent set of approaches, given that microbial signal transductionemploys principles distinct from metazoan signaling) Indeed, optogeneticcontrol of well-defined biochemical signaling pathways was recentlyachieved both in cultured cells and in freely moving mammals, using theoptoXR method for optical control of specified G protein-coupledreceptor signaling (Airan et al., 2009). A photoactivatable adenylylcyclase has been studied from Euglena, although with high dark activitythat limits in vivo application (Schroder-Lang et al., 2007), andsubsequent work on light-sensitive PAS or LOV domains (Levskaya et al.,2009; Wu et al., 2009) may open up new ways to control protein-proteinassociation if these approaches can be made to operate in livinganimals.

We have found that in the nervous system, optogenetic tools can beapplied to probe the neural circuit underpinnings of informationtransmission, oscillations, locomotion, awakening, and reward, as wellas to probe the operation of neural circuits important in a number ofbrain diseases including Parkinson's disease and epilepsy (Adamantidiset al., 2007; Airan et al., 2009; Cardin et al., 2009; Gradinaru et al.,2009; Sohal et al., 2009; Tonnesen et al., 2009; Tsai et al., 2009).Moreover, results thus far point to substantial versatility of theoptogenetic approach across animal species (Adamantidis et al., 2007;Airan et al., 2009; Aravanis et al., 2007; Arenkiel et al., 2007; Bi etal., 2006; Boyden et al., 2005; Chow et al., 2010; Douglass et al.,2008; Gradinaru et al., 2008, 2009; Hägglund et al., 2010; Han et al.,2009a; Huber et al., 2008; Hwang et al., 2007; Ishizuka et al., 2006; Liet al., 2005; Nagel et al., 2003, 2005; Petreanu et al., 2007, 2009;Tsai et al., 2009; Wang et al., 2007; Zhang et al., 2006, 2007a; Zhangand Oertner, 2007; Zhao et al., 2008). Together with fiberoptic(Adamantidis et al., 2007; Aravanis et al., 2007) and integratedfiberoptic-electrode “optrode” assemblies (Gradinaru et al., 2007), evencells located deep within large, dense organs can be readily accessedand interrogated in freely moving mammals. The additional resourcesdefined here arise from the application of molecular, cellular, andgenomic strategies to enable expansion of the capabilities of opticalcontrol, and, as this toolbox rapidly grows, optogenetics may come toplay an increasingly potent and versatile role in intact-systems biologyfor the fast control of defined cells within functioning tissues.

Experimental Procedures

Constructs

All NpHR variants were produced by PCR amplification of the NpHR-EYFPconstruct previously published (Zhang et al., 2007b). All opsinsdescribed here have been optimized for mammalian expression by changingeach gene's codon usage to conform to human codon usage distribution.Updated maps and vectors are available and freely distributed from theDeisseroth laboratory (affiliated with Stanford University, Stanford,Calif.) and described at 2010 Scientific American, “Method of the Year,”December 2010 (http://www.optogenetics.org/), the contents of which arehereby incorporated by reference in their entirety.

The amino acid sequence of GtR3 without the signal peptide sequence(SEQ ID NO: 1) A S S F G K A L L E F V F I V F A C I TL L L G I N A A K S K A A S R V L F P AT F V T G I A S I A Y F S M A S G G G WV I A P D C R Q L F V A R Y L D W L I TT P L L L I D L G L V A G V S R W D I MA L C L S D V L M I A T G A F G S L T VG N V K W V W W F F G M C W F L H I I FA L G K S W A E A A K A K G G D S A S VY S K I A G I T V I T W F C Y P V V W VF A E G F G N F S V T F E V L I Y G V LD V I S K A V F G L I L M S G A A T G Y E S IThe amino acid sequence of GtR3 with thesignal peptide sequence from ChR2 (SEQ ID NO: 5)M D Y G G A L S A V G R E L L F V T N PV V V N G S V L V P E D Q C Y C A G W IE S R G T N G A S S F G K A L L E F V FI V F A C I T L L L G I N A A K S K A AS R V L F P A T F V T G I A S I A Y F SM A S G G G W V I A P D C R Q L F V A RY L D W L I T T P L L L I D L G L V A GV S R W D I M A L C L S D V L M I A T GA F G S L T V G N V K W V W W F F G M CW F L H I I F A L G K S W A E A A K A KG G D S A S V Y S K I A G I T V I T W FC Y P V V W V F A E G F G N F S V T F EV L I Y G V L D V I S K A V F G L I L M S G A A T G Y E S IThe amino acid sequence of DChR (SEQ ID NO: 2)M R R R E S Q L A Y L C L F V L I A G WA P R L T E S A P D L A E R R P P S E RN T P Y A N I K K V P N I T E P N A N VQ L D G W A L Y Q D F Y Y L A G S D K EW V V G P S D Q C Y C R A W S K S H G TD R E G E A A V V W A Y I V F A I C I VQ L V Y F M F A A W K A T V G W E E V YV N I I E L V H I A L V I W V E F D K PA M L Y L N D G Q M V P W L R Y S A W LL S C P V I L I H L S N L T G L K G D YS K R T M G L L V S D I G T I V F G T SA A L A P P N H V K V I L F T I G L L YG L F T F F T A A K V Y I E A Y H T V PK G Q C R N L V R A M A W T Y F V S W AM F P I L F I L G R E G F G H I T Y F GS S I G H F I L E I F S K N L W S L L GH G L R Y R I R Q H I I I H G N L T K KN K I N I A G D N V E V E E Y V D S N D K D S D VThe amino acid sequence of NpHR (SEQ ID NO: 3)M T E T L P P V T E S A V A L Q A E V TQ R E L F E F V L N D P L L A S S L Y IN I A L A G L S I L L F V F M T R G L DD P R A K L I A V S T I L V P V V S I AS Y T G L A S G L T I S V L E M P A G HF A E G S S V M L G G E E V D G V V T MW G R Y L T W A L S T P M I L L A L G LL A G S N A T K L F T A I T F D I A M CV T G L A A A L T T S S H L M R W F W YA I S C A C F L V V L Y I L L V E W A QD A K A A G T A D M F N T L K L L T V VM W L G Y P I V W A L G V E G I A V L PV G V T S W G Y S F L D I V A K Y I F AF L L L N Y L T S N E S V V S G S I L D V P S A S G T P A D DThe amino acid sequence of BR (SEQ ID NO: 4)M L E L L P T A V E G V S Q A Q I T G RP E W I W L A L G T A L M G L G T L Y FL V K G M G V S D P D A K K F Y A I T TL V P A I A F T M Y L S M L L G Y G L TM V P F G G E Q N P I Y W A R Y A D W LF T T P L L L L D L A L L V D A D Q G TI L A L V G A D G I M I G T G L V G A LT K V Y S Y R F V W W A I S T A A M L YI L Y V L F F G F T S K A E S M R P E VA S T F K V L R N V T V V L W S A Y P VV W L I G S E G A G I V P L N I E T L LF M V L D V S A K V G F G L I L L R S RA I F G E A E A P E P S A G D G A A A T S D

Hippocampal Cultures

Primary cultured hippocampal neurons were prepared from P0 Spague-Dawleyrat pups. The CA1 and CA3 regions were isolated, digested with 0.4 mg/mlpapain (Worthington, Lakewood, N.J.), and plated onto glass coverslipsprecoated with 1:30 Matrigel (Beckton Dickinson Labware, Bedford, Mass.)at a density of 65,000/cm². Hippocampal cultures grown on coverslipswere transfected or transduced at 4 days in vitro (DIV) withtiter-matched viruses for all constructs (final dilution 10⁴ infectiousunits (i.u.)/ml in neuronal growth medium). Whole-cell patch clamprecordings were performed as previously described (Zhang et al., 2007b).Primary hippocampal cultures were infected at 4 DIV with titer-matchedvirus (final dilution 104 i.u./ml in neuronal growth medium). At 14 DIV,cultures were fixed for 30 min with ice-cold 4% paraformaldehyde andthen permeabilized for 30 min with 0.4% saponin in 2% normal donkeyserum (NDS). Primary antibody incubations were performed overnight at 4°C.; Cy3-conjugated secondary antibodies (Jackson Laboratories, WestGrove, Pa.) were applied in 2% NDS for 1 hr at room temperature. Imageswere obtained on a Leica confocal microscope with a 63×/1.4 NA oilobjective.

Stereotactic Injection into the Rodent Brain and Optrode Recordings

Adult mice and Long-Evans rats were housed according to the approvedprotocols at Stanford. All surgeries were performed under asepticconditions. The animals were anesthetized with intraperitonealinjections of a ketamine (80 mg/kg)/xylazine (15-20 mg/kg) cocktail(Sigma). The virus was delivered via a 10 μl syringe and a thin 34 gaugemetal needle; the injection volume and flow rate (1 μl at 0.1 μl/min)were controlled with an injection pump from World Precision Instruments(Sarasota, Fla.). For validation of opsin functionality, simultaneousoptical stimulation and electrical recording in living rodents wasconducted as described previously (Gradinaru et al., 2007) with anoptrode composed of an extracellular tungsten electrode (1 MΩ, ˜125 μm)attached to an optical fiber (˜200 μm) with the tip of the electrodedeeper (˜0.4 mm) than the tip of the fiber to ensure illumination of therecorded neurons. The optical fiber was coupled to a 473 nm (for ChR2)or 560 nm (for eNpHR3.0) laser diode (10 mW fiber output) fromCrystaLaser. Optrode recordings were conducted in rodents anesthetizedwith 1.5% isoflurane, and the optrode was placed through smallcraniotomies created above target regions. pClamp 10 and a Digidata1322A board were used to both collect data and generate light pulsesthrough the fiber. The recorded signal was band-pass filtered at 300 Hzlow/5 kHz high (1800 Microelectrode AC Amplifier).

Tissue Slice Preparation

For preparation of brain slices, mice or rats were sacrificed 4 to 5weeks after viral injection. Rodents were perfused with 20 ml ice-coldPBS, followed by 20 ml 4% paraformaldehyde. The brains were then fixedovernight in 4% paraformaldehyde and transferred to 30% sucrose solutionfor 2 days. Brains were frozen and coronal slices (40 μm) were preparedwith a Leica SM2000R cryostat and preserved in 4° C. in cryoprotectant(25% glycerol and 30% ethylene glycol in PBS). Slices (DAPI stain1:50,000) were mounted with PVA-DABCO on microscope slides, and singleconfocal optical sections (e.g., through dorsal CA1 region, ˜1-2.5 mmposterior to bregma or the dorsal subiculum, 2.7-3 mm posterior tobregma) were acquired using a 10× air and 40×/1.4 NA oil objectives on aLeica confocal microscope.

Extended Experimental Procedures

Opsin Sources

All opsins described here have been optimized for mammalian expressionby changing each gene's codon usage to conform to human codon usagedistribution(http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=9606). TheGenBank accession code for the original AR, BR, and GtR3 sequences areDQ074124, M11720, and EG722553.

DNA Constructs

All NpHR variants were produced by PCR amplification of the NpHR-EYFPconstruct previously published (Zhang et al., 2007b) and cloned in-frameinto the AgeI and EcoRI restriction sites of a lentivirus carrying theCaMKIIα or Synapsin-1 promoters according to standard molecular biologyprotocols. A similar strategy was used for BR and AR. GtR3 wasidentified through genomic searches. All opsins described here have beenoptimized for mammalian expression by changing each gene's codon usageto conform to human codon usage distribution(http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=9606), andthe optimized sequence was custom synthesized (DNA2.0, Inc., Menlo Park,Calif.). The GenBank accession codes for the original AR, BR, and GtR3sequences are DQ074124, M11720, and EG722553.pAAV-EF1a-mCherry-IRES-WGA-Cre vector was constructed using standardmolecular biology protocols. Codons for the WGA and Cre genes wereoptimized for expression in mammalian cells. The genes were synthesizedby DNA2.0 (Menlo Park, Calif.). Cre was fused in-frame to the C-term ofWGA, which in turn was fused to IRES. The mCherry-IRES-WGA-Crebicistronic expression cassette was designed using the EMCV IRESsequence. The pAAV-EF1a plasmid backbone is the same as describedpreviously (Sohal et al., 2009; Tsai et al., 2009).pAAV-hSyn-eNpHR3.0-EFYP-P2A-ChR2H134R-mCherry was constructed with a120-mer primer(5′caagttctgctacgagaacgaggtgggctccggagccacgaacttctctctgttaaagcaagcaggagacgtggaagaaaaccccggtcccatggactatggcggcgctttgtctgccg 3′) that containedthe p2A region with the ER export sequence at the 5′ end and 20 bases ofthe start of hChR2 at the 3′ end. First, the ChR2(H134R)-mCherryfragment was amplified using the 120-mer as the forward primer and5′-atatcgaattctcattacttgtacagctcgt-3′ as the reverse primer. Second,this amplified product was used as a reverse primer along with theforward primer 5′-ccggatccccgggtaccggtaggccaccatgacagagaccctgcct-3′ tofuse eNpHR 3.0-EYFP to ChR2 (H134R)-mCherry with the p2A regioninterposed. The 3.4 Kb fragment was then purified and cloned into theBamHI and EcoRI sites of the pAAV-hSyn vector. All constructs were fullysequenced for accuracy of cloning; updated maps are available online athttp://www.optogenetics.org, the contents of which are herebyincorporated by reference in their entirety.

Lentivirus Preparation and Titering

Lentiviruses for cultured neuron infection and for in vivo injectionwere produced as previously described (Zhang et al., 2007b). Viraltitering was performed in HEK293 cells that were grown in 24-well platesand inoculated with 5-fold serial dilutions in the presence of polybrene(8 μg/ml). After 4 days, cultures were resuspended in PBS and sorted forEYFP fluorescence on a FACScan flow cytometer (collecting 20,000 eventsper sample) followed by analysis using FlowJo software (Ashland, Oreg.).The titer of the virus was determined as follows: [(% of infectedcells)×(total number of cells in well)×(dilution factor)]/(volume ofinoculum added to cells)=infectious units/ml. The titer of viruses forculture infection was 10⁵ i.u./ml. The titer of concentrated virus forin vivo injection was 10¹⁰ i.u./ml.

Hippocampal Cultures

Primary cultured hippocampal neurons were prepared from P0 Spague-Dawleyrat pups. The CA1 and CA3 regions were isolated, digested with 0.4 mg/mLpapain (Worthington, Lakewood, N.J.), and plated onto glass coverslipsprecoated with 1:30 Matrigel (Beckton Dickinson Labware, Bedford, Mass.)at a density of 65,000/cm². Cultures were maintained in a 5% CO₂ humidincubator with Neurobasal-A medium (Invitrogen Carlsbad, Calif.)containing 1.25% FBS (Hyclone, Logan, Utah), 4% B-27 supplement (GIBCO,Grand Island, N.Y.), 2 mM Glutamax (GIBCO), and FUDR (2 mg/ml, Sigma,St. Louis, Mo.).

Calcium Phosphate Transfection

6-10 div hippocampal neurons were grown at 65,000 cells/well in a24-well plate. DNA/CaC CI₂ mix for each well: 1.5-3 μg DNA (QIAGENendotoxin-free preparation) +1.875 μ12M CaC CI₂ (final Ca²⁺concentration 250 mM) in 15 μl total H₂O. To DNA/CaCl₂ was added 15 μlof 2× HEPES-buffered saline (pH 7.05), and the final volume was mixedwell by pipetting. After 20 min at RT, the 30 μl DNA/CaCI2₂/HBS mixturewas dropped into each well (from which the growth medium had beentemporarily removed and replaced with 400 μl warm MEM) and transfectionallowed to proceed at 37 C for 45-60 min. Each well was then washed with3×1 mL warm MEM and the growth medium replaced. Opsin expression wasgenerally observed within 20-24 hr.

Electrophysiology Hippocampal cultures grown on coverslips weretransduced at 4 div with titer-matched viruses for all constructs (finaldilution 10⁴ i.u./ml in neuronal growth medium) and allowed to expressfor one week. Whole-cell patch clamp recordings were performed aspreviously described (intracellular solution: 129 mM K-gluconate, 10 mMHEPES, 10 mM KCl, 4 mM MgATP, 0.3 mM Na₃GTP, titrated to pH 7.2;extracellular Tyrode: 125 mM NaCl, 2 mM KCl, 3 mM CaCl2, 1 mM MgCl₂, 30mM glucose, and 25 mM HEPES, titrated to pH 7.3). For voltage clamprecordings cells were held at −70 mV. Light in the visible range wasdelivered from a 300 W DG-4 lamp (Sutter Instruments, Novato, Calif.)through filters of different wavelength selectivity (Semrock, Rochester,N.Y.) and a Leica 40×/0.8NA water objective. Filters, except for powerspectra, (given here as wavelength in nm/bandwidth in nm/output power inmW/mm²) were: 406/15/3; 472/30/18.5; 560/14/7; 589/15/7.5; 593/40/15.5;630/20/3.5. Far-red and near-infrared light delivery: light (7 mW/mm2)for 660 nm inhibition was delivered using a light emitting diode and a40×/.8 NA water objective. Light (7 mW/mm²) for 680 nm inhibition wasdelivered using the X-Cite 120 W halogen light source through a 680±13nm filter and a 40×/0.8 NA water objective. Light delivery for eNPAC,ChR2(H134R), and eNpHR3.0 power spectra was delivered from a 300 W DG-4lamp fitted with a Lambda 10-3 filter wheel (Sutter Instruments) with a10-position wheel for 25-mm filters of different wavelengths and a40×/0.8NA water objective. Filters (given here as wavelength innm/bandwidth in nm/output power in mW/mm²) were: 387/10/3.5; 406/15/3.5;427/10/4.0; 445/20/4.0; 470/22/4.0; 494/20/4.5; 520/15/4.5; 542/27/5.0;560/20/5.0; 590/20/3.5; 630/20/3.5. For FIGS. 1, 3A-3D, and 4 and FigureS2 (see, Table 5 below), confocal images and whole-cell patch clamp dataare from cultured hippocampal neurons either transfected (confocal data)or transduced (patch data) with lentiviral NpHR, BR, GtR3 and AR-basedconstructs, and allowed to express for one week. Expression was drivenby the human Synapsin I promoter and visualized by fusion to EYFP.

Table 5 shows additional trafficking-enhanced tools for blue inhibition,and a new opsin sequence: G. theta rhodopsin-3 or GtR3.

TABLE 5

New opsin sequence: G. theta rhodopsin-3 or GtR3. The EST sequenceincluded all seven transmembrane helices; the 5′ amino acid sequence wasprovided from ChR2 (transmembrane motifs: bars; conserved residues:highlighted; truncation site for peptide: *; signal peptide providedfrom ChR2: gray.

Immunohistochemistry Primary hippocampal cultures were infected at 4 divwith titer matched virus (final dilution 10⁴ i.u./ml in neuronal growthmedium). At 14 div cultures were fixed for 30 min with ice-cold 4%paraformaldehyde and then permeabilized for 30 min with 0.4% saponin in2% normal donkey serum (NDS). Primary antibody incubations wereperformed overnight at 4° C. using a monoclonal marker of endoplasmicreticulum recognizing endogenous ER-resident proteins containing theKDEL retention signal (KDEL 1:200, Abcam, Cambridge, Mass.).Cy3-conjugated secondary antibodies (Jackson Laboratories, West Grove,Pa.) were applied in 2% NDS for 1 hr at room temperature. Images wereobtained on a Leica confocal microscope using a 63×/1.4NA oil objective.

Stereotactic Injection into the Rodent Brain Adult mice and Long-Evansrats were housed according to the approved protocols at Stanford. Allsurgeries were performed under aseptic conditions. The animals wereanesthetized with intraperitoneal injections of a ketamine (80mg/kg)/xylazine (15-20 mg/kg) cocktail (Sigma). The head was placed in astereotactic apparatus (Kopf Instruments, Tujunga, Calif.; Olympusstereomicroscope). Ophthalmic ointment was applied to prevent eyedrying. A midline scalp incision was made and a small craniotomy wasperformed using a drill mounted on the stereotactic apparatus (FineScience Tools, Foster City, Calif.). The virus was delivered using a 10μl syringe and a thin 34 gauge metal needle; the injection volume andflow rate (1 μl at 0.1 CI₂l/min) was controlled with an injection pumpfrom World Precision Instruments (Sarasota, Fla.). After injection theneedle was left in place for 5 additional minutes and then slowlywithdrawn. The skin was glued back with Vetbond tissue adhesive. Theanimal was kept on a heating pad until it recovered from anesthesia.Buprenorphine (0.03 mg/kg) was given subcutaneously following thesurgical procedure to minimize discomfort. For the experiment in FIG.2A, to cover a large area in dorsal CA1, 1 μl of concentrated lentivirus(10¹⁰ i.u./ml) carrying eNpHR3.1 (a shorter form of eNpHR3.0 with theN-terminal signal peptide, the first 17 amino acids of original NpHR,removed) under the CaMKIIα promoter was microinjected into 2 sites ineach hippocampus (site one: anteroposterior −1.5 mm from bregma;lateral, ±1 mm; ventral, 1.5 mm; site two: AP, −2.5 mm from bregma;lateral, ±2 mm; ventral, 1.5 mm) For FIGS. 2D and 2E, two differentadeno-associated viruses (AAVs) (virus titer 2×10¹² g.c./mL), werestereotactically injected during the same surgery with an injectionspeed of 0.15 ul/min High-titer (2×10¹² g.c./mL) AAV was produced by theUNC VectorCore. For FIG. 2D, double-floxed cre-dependent AAV5 carryingeNpHR3.0-EYFP (AAV5-Ef1a-DIO-eNpHR3.0-EYFP) was injected into M1, andAAV2-Ef1α-mCherry-IRESWGA-Cre was injected into 51 of adult Long-Evansrats. 1 μl of virus was delivered at five different sites defined by thefollowing coordinates: M1 injection I: AP, +1 mm from bregma; lateral,1.5 mm; ventral, 2 mm; M1 injection II: AP, +2 mm; lateral, 1.5 mm;ventral, 2 mm; 51 injection I: AP, −0.3 mm; lateral, 3.4 mm; ventral, 2mm; S1 injection II: AP, −1.3 mm; lateral, 3 mm; ventral, 2 mm; S1injection III: AP, −2.12 mm; lateral, 3 mm; ventral, 2 mm. For FIG. 2E,1 μl of virus was injected bilaterally into the dentate gyrus (DG) ofadult BL6 mice. AAV8-EF1a-DIO-ChR2-EYFP was injected in the right DG andof AAV2-EF1a-mCherry-IRES-WGA-Cre was injected in the left DG with thefollowing coordinates: AP, −2.1 from bregma; lateral, ±1.05 mm; ventral,2.1 mm.

In Vivo Optrode Recordings

To validate opsin functionality in the WGA-Cre system simultaneousoptical stimulation and electrical recording in living rodents wasconducted as described previously (Gradinaru et al., 2007) using anoptrode composed of an extracellular tungsten electrode (1 MΩ, ˜125 um)attached to an optical fiber (˜200 μm) with the tip of the electrodedeeper (˜0.4 mm) than the tip of the fiber to ensure illumination of therecorded neurons. The optical fiber was coupled to a 473 nm (for ChR2)or 560 nm (for eNpHR3.0) laser diode (10 mW fiber output) fromCrystaLaser. Optrode recordings were conducted in rodents anesthetizedwith 1.5% isoflurane and the optrode was placed through smallcraniotomies created above target regions. pClamp 10 and a Digidata1322A board were used to both collect data and generate light pulsesthrough the fiber. The recorded signal was band pass filtered at 300 Hzlow/5 kHz high (1800 Microelectrode AC Amplifier). For precise placementof the fiber/electrode pair, stereotactic instrumentation was used.

Tissue Slice Preparation

For preparation of brain slices, mice or rats were sacrificed 4 to 5weeks after viral injection. Rodents were perfused with 20 ml ofice-cold PBS, followed by 20 ml of 4% paraformaldehyde. The brains werethen fixed overnight in 4% paraformaldehyde, and transferred to 30%sucrose solution for 2 days. Brains were frozen and coronal slices (40μm) were prepared using a Leica SM2000R cryostat, and preserved in 4° C.in cryoprotectant (25% glycerol, 30% ethylene glycol, in PBS). Slices(DAPI stain 1:50,000) were mounted with PVA-DABCO on microscope slides,and single confocal optical sections (e.g., through dorsal CA1 region,˜1-2.5 mm posterior to bregma or the dorsal subiculum, 2.7-3 mmposterior to bregma) were acquired using a 10× air and 40×/1.4NA oilobjectives on a Leica confocal microscope.

For further details and discussion of the above-noted embodiments,reference can be made to “Molecular and Cellular Approaches forDiversifying and Extending Optogenetics” by Viviana Gradinaru et al.,Cell 141, 154-165, Apr. 2, 2010, which is fully incorporated herein byreference.

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All references, publications, and patent applications disclosed hereinare hereby incorporated by reference in their entirety.

The various embodiments described above are provided by way ofillustration only and should not be construed to limit the invention.Based on the above discussion and illustrations, those skilled in theart will readily recognize that various modifications and changes may bemade to the present invention without strictly following the exemplaryembodiments and applications illustrated and described herein. Forinstance, such changes may include the use of digital logic ormicroprocessors to control the emitted light. Such modifications andchanges do not depart from the true spirit and scope of the presentinvention, which is set forth in the following claims. As discussedabove, specific applications and background details relative to thepresent invention are discussed above, in the description below andthroughout the references cited herein. The embodiments in theAppendices may be implemented in connection with one or more of theabove-described embodiments and implementations, as well as with thoseshown in the figures and described below. Reference may be made to theAppendices (A, B and C) which were filed in the underlying provisionalapplication and incorporated herein by reference.

1.-77. (canceled)
 78. A system comprising: a) a delivery devicecomprising a polynucleotide that comprises a nucleotide sequenceencoding a light-activated polypeptide, wherein the light-activatedpolypeptide comprises, from N-terminus to C-terminus: i) a core aminoacid sequence that is at least 95% identical to the sequence shown inSEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:4; ii) anendoplasmic reticulum (ER) export signal; and iii) a membranetrafficking signal; b) a light source; and c) a control device thatcontrols generation of light by the light source.
 79. The system ofclaim 78, wherein the control circuit is arranged to respond to anexternal signal.
 80. The system of claim 78, wherein the control circuitcomprises one or more of a rectifier circuit, a battery, a pulse timer,and a comparator circuit.
 81. The system of claim 78, wherein thecontrol circuit comprises a integrated circuit.
 82. The system of claim78, wherein all or a part of the system is implantable.
 83. The systemof claim 78, wherein the polynucleotide is present in a matrix.
 84. Thesystem of claim 78, wherein the polynucleotide is present in a gel. 85.The system of claim 78, wherein the light source comprises alight-emitting diode.
 86. The system of claim 78, wherein the lightsource comprises a fiber optic.
 87. The system of claim 78, wherein theER export signal comprises the amino acid sequence
 88. The system ofclaim 78, wherein the membrane trafficking signal comprises the aminoacid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:11).
 89. The system ofclaim 78, wherein the core amino acid sequence is at least 95% identicalto amino acids 25-265 of SEQ ID NO:2.
 90. The system of claim 78,wherein the core amino acid sequence is at least 95% identical to SEQ IDNO:3.
 91. The system of claim 78, wherein the core amino acid sequenceis at least 95% identical to SEQ ID NO:4.
 92. The system of claim 78,wherein the light-activated protein-encoding nucleotide sequence isoperably linked to a promoter.
 93. The system of claim 92, wherein thepromoter is a CaMKIIa promoter.
 94. The system of claim 92, wherein thepromoter is a synapsin I promoter.
 95. The system of claim 78, whereinthe polynucleotide is in an expression vector.
 96. The system of claim95, wherein the expression vector is a viral vector.
 97. The system ofclaim 96, wherein the viral vector is an adenoassociated virus vector, aretroviral vector, an adenoviral vector, a herpes simplex virus vector,or a lentiviral vector.